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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: Combined DNA damage and/or repair and chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 489 (Alkaline Comet Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: combined Comet and Micronucleus assay

Test material

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar rats (outbred, SPF-Quality) are used as test system. These rats are recommended by international guidelines (e.g. EPA, FDA, OECD, EC).
Sex:
male
Details on test animals or test system and environmental conditions:
ANIMALS
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.
Young adult animals were selected (6-7 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 3 and in the main study 30. In the main study 5 male rats were treated per sampling time in each treatment group. Six additional animals were used for blood sampling for bioanalysis. These animals were treated with vehicle or the highest test item concentration (3 animals per group).
The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. The body weights of the rats at the start of the treatment with Sa 57 were within 20% of the sex mean. The mean body weight was 151 (± 7.4 g) and the range was 135 – 186 g. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups. The acclimatization period was at least 6 days before the start of treatment under laboratory conditions. 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.


CONDITIONS
A controlled environment was maintained in the room with optimal conditions of approximately 10 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 19.2 – 21.8°C), a relative humidity of 40 - 70% (actual range: 37 - 68%) and a 12 hour light/12 hour dark cycle. Due to e.g. cleaning procedures, temporary deviations from the humidity (maximum 3%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Accommodation: Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Diet: The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.

Water: The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands)

No correction was made for the purity/composition of the test item. Sa 57 was suspended (2000 and 1000 mg/kg group) or dissolved (500 mg/kg group) in corn oil. The specific gravity of corn oil is 0.9 g/mL. Sa 57 concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension or to dissolve the test item (for a maximum of 17 minutes at 28ºC in the main study). Sa 57 concentrations were dosed within 4 hours after preparation.
Details on exposure:
The rats were dosed for three consecutive days (once daily) using an oral intubation with a plastic gavage needle of a high, an intermediate and a low dose of Sa 57. The rats were dosed twice with the positive control EMS and once with CP. A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat).

The first dose of the test item and vehicle was administered at t = 0 h. The second and third dose will be administered at t=24 h and t= 45, respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 and t=45. The animals were sacrificed at approximately t = 48 h by abdominal aorta bleeding under isoflurane anesthesia.

Animals were checked for mortality at least twice a day. The time of death was recorded as precisely as possible. The systemic toxic signs were recorded at least once a day from start of treatment onwards. All signs were recorded. The animals were weighed just prior to dosing.
Duration of treatment / exposure:
3 d
Frequency of treatment:
once daily for three consecutive days
Post exposure period:
- Dose-range Finding study: 1-4 days (mortality and physical condition were recorded at least once a day)
- Main study: Sacrifice on day 3
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control; Group A and A'
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Group B and B'
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group C
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group D
No. of animals per sex per dose:
5 (plus 3 additional animals for vehicle control and highest dose group)
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control for the micronucleus test was Cyclophosphamide (CP; CAS no. 50-18-0) at 20 mg/kg body weight dissolved in physiological saline. The stock solutions of CP were stored in aliquots at ≤ -15°C in the dark and one sample was thawed immediately before use. The route of administration was oral and the dosing volume was 10 mL/kg body weight.

The positive control for the Alkaline Comet test was Ethyl Methanesulfonate (EMS; CAS no. 62-50-0) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 3 hours after preparation and the route of administration was oral. The dosing volume was 10 mL/kg body weight.

Examinations

Tissues and cell types examined:
The bone marrow was examined for micronuclei while the Comet part of the combined assay was conducted with liver tissue and (glandular) stomach cells.
Blood was collected from the six satellite animals (high dose group and vehicle control group) for bioanalysis.
Details of tissue and slide preparation:
BONE MARROW SMEARS FOR MICRONUCLEI
The supernatant was removed with a Pasteur pipette. Approximately 500 µL serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, the Netherlands). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands)

COMET SLIDES
To 20 µL of the cell suspension, 280 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and
50 µL was layered on a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 10-42 minutes in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.
The cells on the slides were overnight (approximately 16-17 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 29-32 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm for liver and at 0.7 Volt/cm for stomach. The electrophoresis was performed for 30 minutes under constant cooling (actual temperature 4.0 °C) for liver and for 20 minutes under constant cooling (actual temperature 4.0 °C) for stomach. After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in absolute ethanol (Merck, Darmstadt, Germany) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

Evaluation criteria:
see "Any other information on materials and methods"
Statistics:
Student's t test; Dunnett's test; ToxRat Professional v 3.2.1

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
micronucleus test
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
comet assay
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

DOSE RANGE FINDING STUDY

In a dose-range finding study 3 male animals were dosed via oral gavage with 2000 mg/kg body weight of SA 57. One animals showed no treatment related clinical signs after the first and second dosing. Within approximately one hour after the third dosing this animal was lethargic and within two hours after the third dosing this animal showed in addition ataxia. The animal appeared recovered within 20-21 hours after the third dosing. The other two animals were lethargic and had diarrhea within approximately 1 hour after the first dosing. The animals appeared recovered within approximately 22 hours after the first dosing. Within approximately 1 hour after the second dosing the animals were lethargic. The animals appeared recovered within approximately 22 hours after the second dosing but the animals did not completely eat the limited amount of food that was provided for them overnight. Within approximately 1 hour after the third dosing the animals were lethargic and showed ataxia. The severity of the effects was generally considered to be slight.

MAIN STUDY

Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg body weight were selected as appropriate doses for the main study.

Mortality and Toxic Signs

The animals of the group treated with 500 mg Sa 57/kg bw and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. The following clinical observations were made in the group treated with 1000 mg Sa 57 /kg bw: The animals showed no treatment related clinical signs after the first and second dosing. Within approximately 1 hour after the third dosing 3 of the 5 animals were lethargic. The other two animals showed no treatment related clinical signs.

The following clinical observations were made in the group treated with 2000 mg Sa 57 /kg bw: Within approximately the first hour after the first dosing all animals were lethargic. In addition one animal showed ataxia. Within approximately one hour after the second dosing all animals were lethargic and showed ataxia. All animals appeared recovered within approximately 19 hours after the second dosing. Within approximately one hour after the third dosing all animals were lethargic and showed ataxia. The severity of the effects was generally considered to be slight.

Micronucleated polychromatic erythrocytes

The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of polychromatic to normochromatic erythrocytes are presented in the table below. The mean number of micronucleated polychromatic erythrocytes scored in Sa 57 treated groups were compared with the corresponding solvent control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Sa 57 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. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

Ratio of polychromatic to normochromatic erythrocytes

The animals of the groups which were treated with Sa 57 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item on erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

 Group  Dose [mg/kg bw]  Mean no. polychromatic erythrocytes  Mean ratio polychrom. / normochrom. erythrocytes
 A vehicle control  0  5.0  0.99
 B Sa 57  2000  2.6  0.88
 C Sa 57  1000  2.4  0.99
 D Sa 57  500  3.4  1.09
 F pos. control CP  20  40.4  0.46

Comet Slide Analysis

After treatment, single cell suspensions from the liver and stomach were prepared. The viability of one single cell suspension per tissue per group was assessed by using trypan blue. The viability of all single suspension was 100%. Comet slides were prepared and analyzed.

- Liver: No statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of Sa 57-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity in liver cells of male vehicle-treated rats was 4.13%. All values were within the historical data control range. EMS, the positive control item, showed a Mean Tail Intensity of 97.96%. EMS treatment induced a statistically significant increase of 23.7-fold (p<0.001 Student’s t test) in the mean Tail Intensity (%) in liver cells of male rats when compared to the vehicle. Hence, the acceptability criteria of the test were met.

- Glandular Stomach: No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Sa 57-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity in stomach cells of male vehicle-treated rats was 43.05%. All values were within the historical data control range. EMS, the positive control item, showed a Mean Tail Intensity of 88.47%. EMS treatment induced a statistically significant increase of 2.1-fold (p<0.001 Student’s t test) in the mean Tail Intensity (%) in stomach cells of male rats when compared to the vehicle. Hence, the acceptability criteria of the test were met. A decrease in Tail Intensity was observed at 1000 mg/kg in glandular stomach. Since this decrease was only observed at 1000 mg/kg and thus not at 500 and 2000 mg/kg (no dose related effect) and also not in liver this decrease is considered biologically not relevant and caused by variation in the data. Moreover a decrease does not indicate a genotoxic effect and overall there is thus no effect on the study conclusion.

 Group  Dose [mg/kg bw]  Tail Intensity [%] liver cells  Tail Intensity [%] stomach cells
 A vehicle control  0  4.13  43.05
 B Sa 57  2000  5.10  41.23
 C Sa 57  1000  4.12  17.26
 D Sa 57  500  2.73  50.18
 E pos. control EMS  200  97.96  88.47

Chemical analysis of dose preparations

The formulations of the second dosing of the main study were analyzed for concentration, homogeneity and stability.

- Concentration: The concentrations analyzed in the formulations of Group D (500 mg/kg bw), Group C (1000 mg/kg bw) and Group B (2000 mg/kg bw) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The mean accuracies were 100, 100 and 102% for Group D, C, B, respectively. No test item was detected in the Group A (vehicle control) formulation.

- Homogeneity: The formulations of Group D (500 mg/kg bw) and Group B (2000 mg/kg bw) were homogeneous (i.e. coefficient of variation ≤ 10%). The homogeneity (coefficient of variation %) was 3.4 and 5.0% for group D and B, respectively.

- Stability: Formulations of Group D (500 mg/kg bw) and Group B (2000 mg/kg bw) were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours (i.e. mean concentrations after storage ± 10%). The relative difference between the mean concentration of the stability samples and six samples at t=0 taken at 10%, 50% and 90% height was -0.91 and -1.2% for group D and B, respectively.

Applicant's summary and conclusion

Conclusions:
It is concluded that the micronucleus test was valid and that Sa 57 is not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 2000 mg/kg under the experimental conditions described in this report. Moreover, the comet assay was valid and Sa 57 does not provoke DNA damage in the Comet assay in liver and glandular stomach cells under the experimental conditions described in this report.
Executive summary:

Sa 57 was tested in a combined Micronucleus and Comet test in the rat.

The objective of the study was to obtain information on the potential genotoxicity of Sa 57 when administered to rats at a maximum required acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in liver and glandular stomach tissue.

The study procedures described in this report are in compliance with the most recent OECD and EC guidelines.

Batch SS-Sa-11074-80 of SA 57 was a yellow viscous liquidwith a purity of 99.3%. The test item was suspended (2000 mg/kg group and 1000 mg/kg group) or dissolved (500 mg/kg group) in corn oil.

In a dose range finding study, 3 male animals were dosed via oral gavage with Sa 57 at 2000 mg/kg/day body weight for three consecutive days. The treatment related clinical signs of the three animals treated with 2000 mg/kg/day were slight and comprised lethargy, ataxia, diarrhea (2/3 animals) and the limited amount of feed provided overnight before dosing was not completely eaten (2/3 animals). Based on the results of the dose range finding study, a dose level of 2000 mg/kg body weight was selected as the highest dose level in the main study (the maximum recommended dose in accordance with current regulatory guidelines).

In the main study, groups of 5 male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg Sa 57 per kg body weight per day for three consecutive days. A positive control group (5 male rats) for the comet assays was dosed once daily for two consecutive days by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group (5 male rats) for the micronucleus assay was dosed once by oral gavage with 20 mg cyclophosphamide (CP) per kg body weight. In addition, blood for bioanalysis of Sa 57 in plasma was collected from 3 satellite animals for the 2000 mg/kg group (highest dose group) and from three satellite animals for the vehicle control group.

 

The animals of the group treated with 500 mg Sa 57/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality. The animals treated with 1000 mg Sa 57 /kg body weight showed no treatment related clinical signs with exception of 3 animals which were lethargic after the third dose. The following clinical observations were made in the group treated with 2000 mg Sa 57 /kg body weight: Within approximately the first hour after the first dosing all animals were lethargic. In addition one animal showed ataxia. Within approximately one hour after the second dosing all animals were lethargic and showed ataxia. All animals appeared recovered within approximately 19 hours after the second dosing. Within approximately one hour after the third dosing all animals were lethargic and showed ataxia.The severity of the effects was generally considered to be slight.

 

Approximately 3-4 hours after the third dose of the vehicle or Sa 57, liver and glandular stomach tissue were collected for the comet assay. The animals were sacrificed by abdominal aorta bleeding under isoflurane anaesthesia. Single cell suspensions were made followed by comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

Bone marrow smears were analysed. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with
Sa 57 compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant 8.1-fold increase (Student's t test p<0.001) in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met. The groups that were treated with Sa 57 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

No statistically significant increase in the mean Tail Intensity (%) was observed in liver and glandular stomach cells of Sa 57-treated male rats at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 4.13%. All values were within the historical data control range. EMS, the positive control item, showed a mean Tail Intensity of 97.96%. EMS treatment induced a statistically significant increase of 23.7-fold (p<0.001 Student’s t test) in the mean Tail Intensity (%) in liver cells of male rats when compared to the vehicle treated rats. Hence, the acceptability criteria of the comet test in liver were met. The mean Tail Intensity (%) in glandular stomach cells of vehicle treated male rats was 43.05%. The positive control EMS, showed a mean Tail Intensity of 88.47% (2.1-fold statistically significant induction; Studentsttest p<0.001). The negative and positive control Tail Intensities were within the historical control data range. Hence, all criteria for an acceptable comet assay in stomach were met.

 

Formulation analysis was performed to determine the accuracy of preparation, homogeneity and stability of the test item in formulations. The concentrations analysed in the formulations of the high dose (2000 mg/kg), mid dose (1000 mg/kg) and low dose (500 mg/kg) were in agreement with target concentrations (i.e. mean concentrations of 102, 100 and 100%, respectively). No test substance was detected in the vehicle control. Overall it was concluded that the accuracy of preparation was acceptable. The formulations were homogeneous (i.e. coefficient of variation <5.0%) and stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

It is concluded that the micronucleus test was valid and that Sa 57 is not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 2000 mg/kg under the experimental conditions described in this report. Moreover, the comet assay was valid and Sa 57 does not provoke DNA damage in the Comet assay in liver and glandular stomach cells under the experimental conditions described in this report.

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