Registration Dossier

Administrative data

Endpoint:
genetic toxicity in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study is planned to be conducted in December 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
The micronucleus test (OECD 474) is suitable to investigate other potential mechanisms of genotoxicity such as clastogenicity and aneuploidy.
The comet assay (OECD 489) could exam tissues at the site of contact. The substance does not need to pass through many organs before reaching targeted organ. In the view of optimal use of animals, ECHA agreed to perform a comet assay combined with a micronucleus test.
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Sex:
male
Details on test animals and environmental conditions:
Rats: Crl:WI(Han) (outbred, SPF-Quality) are used as test system. These rats are recommended by international guidelines (e.g. OECD and EC). The animals are 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 Comet main study, 5 male rats were treated in each Everzol Orange ED-G Crude and the vehicle treatment group.

The positive controls EMS and CP were tested in 5 male animals.
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 Everzol Orange ED-G Crude were within 20% of the sex mean. The mean body weights were 148 ± 6.2 g and the range 137 – 159 g

The mean body weights were 145 ± 7.8 g (range 130 – 165 g) at the start of treatment with the positive control EMS. 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.

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Everzol Orange ED-G Crude was suspended in Milli-Ro water. The specific gravity of Milli-Ro water is 1.0 g/mL. Everzol Orange ED-G Crude concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. Everzol Orange ED-G Crude concentrations were dosed within 4 hours after preparation.
Details on exposure:
The rats were dosed for three consecutive days (twice daily) with the test item and vehicle by oral gavage (oral intubation with a plastic gavage needle). The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use. The rats were dosed twice with the positive control EMS and once with CP.

The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h (± 2 h) and t=45 h (± 2 h), respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 (± 2 h) and t=45 (± 2 h). The animals were sacrificed at t = 48 h. The doses were split into two consecutive doses with a 2-3-hour interval for the highest dose group and negative control dose group.

The dosing volume was 10 mL/kg body weight.
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Control animals:
yes, concurrent vehicle
yes, historical
no
Positive control(s):
The positive control for the micronucleus test was Cyclophosphamide (CP; CAS no. 50-18-0; Baxter B.V., Utrecht, The Netherlands) 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 consistent with those of the test item. 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; Sigma Aldrich, Steinheim, Germany) 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

Details of tissue and slide preparation:
Preparation of bone marrow smears for the micronucleus test
- Preparation of bone marrow smears
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. Two slides were prepared per animal.

- Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide
stainer (Miles, Bayer Nederland B.V.). 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 (Leica Microsystems B.V.,
Rijswijk, The Netherlands)

- Analysis of the bone marrow smears for micronuclei
To prevent bias, all slides were randomly coded before examination. An adhesive label with study
identification number and code was stuck over the marked slide. At first the slides were screened at a
magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread,
undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of
micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes
(with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was
determined by counting and differentiating at least the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were
calculated. Parts on the slides that contained mast cells that might interfere with the scoring of
micronucleated polychromatic erythrocytes were not used for scoring.


Preparation of Comet slides
- Determination of cell viability
The viability of the cells after isolation was determined by manually counting the number of viable cells
using trypane blue staining. One animal per group was checked.

- Preparation of slides
To 20 µL of the cell suspension, 180 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 10-15 minutes in the refrigerator in the dark until a clear ring appears at the edge of the
Comet slide area.

- Lysis, electrophoresis and staining of the slides
The cells on the slides were overnight (approximately 17 h) immersed in prechilled lysis solution
(Trevigen) in the refrigerator. After this incubation period, the slides were immersed/rinsed in
neutralization buffer (0.4M Tris-HCl pH 7.4) for approximately 5 minutes. The slides were then placed
in freshly prepared alkaline solution for 30 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. The electrophoresis was performed for 30 minutes under constant
cooling (actual temperature 5.5 – 7.0°C). After completion of electrophoresis, the slides were
immersed/rinsed in the neutralization buffer. The slides were subsequently immersed for 5 minutes in
70% ethanol 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.

- Comet scoring
To prevent bias, slides were randomly coded before examination of the Comets. An adhesive label
with study identification number and code were placed over the marked slide. The slides were
examined with a fluorescence microscope connected to a Comet Assay IV image analysis system
(Perceptive instruments Ltd, Suffolk, United Kingdom).

One hundred fifty Comets per slide (50 comets of each replicate LMAgarose circle) were examined.
On a few slides the number of cells was limited, therefore an agarose circle from the second backup
slide was used for scoring.

The following criteria for scoring of Comets were used:
- Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
- Cells that showed overlap or were not sharp were not scored.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
negative
Remarks:
Micronucleated polychromatic erythrocytes
Toxicity:
no effects
Remarks:
The animals of the all groups treated with 500, 1000 and 2000 mg Everzol Orange ED-G Crude/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
Comet slide analysis
Toxicity:
no effects
Remarks:
The animals of the all groups treated with 500, 1000 and 2000 mg Everzol Orange ED-G Crude/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Micronucleated polychromatic erythrocytes
The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of
polychromatic to normochromatic erythrocytes are presented in Table 2 (APPENDIX 1 ). The individual
data are described in Table 6 (APPENDIX 1 ). The mean number of micronucleated polychromatic
erythrocytes scored in Everzol Orange ED-G Crude 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 Everzol Orange ED-G Crude 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 (APPENDIX 8).
Cyclophosphamide, the positive control item, induced a statistically significant 46-fold increase in the
number of micronucleated polychromatic erythrocytes (APPENDIX 4). Hence, the acceptability criteria
of the test were met

- Ratio polychromatic to normochromatic erythrocytes
The animals of the groups, which were treated with Everzol Orange ED-G Crude showed no decrease
in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of
this test item on the 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.


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% (Table 3; APPENDIX 1).

Comet slides were prepared and analysed. An overview of the mean Tail Intensity is presented in
Table 4 - 5 and Figure 1 - 2 (APPENDIX 1). The detailed data of the treatment groups is presented in
APPENDIX 2. The detailed data of the individual rats and slides is presented in APPENDIX 6-7. The
historical data is presented in APPENDIX 8.

- Liver
A statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of Everzol
Orange ED-G Crude-treated male animals at 1000 and 2000 mg/kg compared to the vehicle treated
animals (p<0.05 Dunnet’s t test). The mean Tail Intensity (%) in liver cells of vehicle treated male rats
was 1.89 ± 0.30% (Mean±SD). The Tail Intensities at 500, 1000 and 2000 mg/kg were 2.52 ± 0.42%,
3.59 ± 1.21 and 2.94 ± 0.49 (Mean ± SD), respectively. Since all values were clearly within the
historical vehicle control data range (4.21 ± 3.08%, Mean ± SD) and no dose related response was
shown, the statistically significant increase observed at 1000 and 2000 mg was considered biologically
not relevant and caused by the low variation in the vehicle control data.

The positive control EMS showed a mean Tail Intensity of 93.73 ± 3.14% (Mean ± SD, 49.6-fold
statistically significant induction; Students t test p<0.001). The negative and positive control Tail
Intensities were within the historical control data range. Hence, all criteria for an acceptable assay
were met.

- Stomach
No statistically significant increase in the mean Tail Intensity was observed in the stomach of
Everzol Orange ED-G Crude-treated male animals at any of the dose levels tested compared to the
vehicle treated animals.

The mean Tail Intensity (%) in stomach cells of vehicle treated male animals was 27.81± 10.65%
(Mean ± SD). The positive control EMS, showed a mean Tail Intensity of 97.93 ± 1.10% (Mean ± SD,
3.5-fold statistically significant induction; Students t test p<0.001; APPENDIX 4). The negative and
positive control Tail Intensities were within the historical control data range. Hence, all criteria for an
acceptable assay were met

Applicant's summary and conclusion

Conclusions:
It is concluded that the micronucleus test was valid and that Everzol Orange ED-G Crude is not
clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of
2000 mg/kg kg (the maximum recommended dose in accordance with current regulatory guidelines)
under the experimental conditions described in this report. Moreover, Everzol Orange ED-G Crude
does not provoke DNA damage in the Comet assay in liver and stomach cells under the experimental
conditions described in this report.
Executive summary:

The compound Everzol Orange ED-G Crude was tested in the combined alkalinein vivoComet assay
in male rats, to evaluate its potential to induce genotoxic effects.


The study procedures described in this report were based on the most recent OECD and EC
guidelines.

Batch F8411502 of Everzol Orange ED-G Crude was a black powder. The test item was suspended in
Milli-Ro water.

Formulation analysis was performed to determine the accuracy of preparation, homogeneity and
stability of the test substance in formulations. The concentrations analysed in the formulations were
considered in agreement with target concentrations (i.e. mean accuracies between 111% and 114%) .
No test item was detected in the vehicle control samples. The formulations were homogeneous (i.e.
coefficient of variation <1.6%) and stable when stored at room temperature under normal laboratory
light conditions for at least 4 hours.

In the dose range finding study 3 males were dosed once daily via oral gavage with 2000 mg Everzol
Orange ED-G Crude per kg body weight for three consecutive days. The animals showed no treatment
related clinical signs or mortality after dosing.

In the main study, groups of 5 male animals were dosed once daily via oral gavage with vehicle or with
2000, 1000 and 500 mg Everzol Orange ED-G Crude per kg body weight for three consecutive days.
A positive control group (5 male rats) for the comet assays was dosed twice 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. The animals showed no treatment related clinical signs or mortality after dosing.

Approximately 3-4 hours after the third dose of the vehicle or Everzol Orange ED-G Crude, liver and
stomach tissue were collected. The animals were sacrificed by abdominal aorta bleeding under
isoflurane anaesthesia and liver and stomach tissue was isolated. Single cell suspensions from the
liver and stomach 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 Everzol Orange
ED-G Crude 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 46-fold increase (Studentsttest p<0.05) 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 Everzol Orange ED-G Crude 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 biologically relevant increase in the mean Tail Intensity (%) was observed in liver and stomach
cells of Everzol Orange ED-G Crude treated male animals compared to the vehicle treated animals.
The mean Tail Intensity (%) in liver and stomach cells of vehicle treated male animals was 1.89
± 0.30% and 27.81± 10.65% (Mean ± SD), respectively. The positive control EMS showed a mean Tail
Intensity of 93.73 ± 3.14% (Mean ± SD, 49.6-fold statistically significant induction; Studentsttest
p<0.001) and 97.93 ± 1.10% (Mean ± SD, 3.5-fold statistically significant induction; Students t test
p<0.00) in liver and stomach, respectively. The negative and positive control Tail Intensities were
within the historical control data range. Hence, all criteria for an acceptable comet assay were met.

 

It is concluded that the micronucleus test was valid and that Everzol Orange ED-G Crude is not

clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of

2000 mg/kg kg (the maximum recommended dose in accordance with current regulatory guidelines)

under the experimental conditions described in this report. Moreover, Everzol Orange ED-G Crude

does not provoke DNA damage in the Comet assay in liver and stomach cells under the experimental

conditions described in this report.