Registration Dossier

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 28, 2019 to June 24, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male
Details on test animals or test system and environmental conditions:
Preliminary toxicity test: Males weighed between 183 g - 190 g.
Females weighed between 176 g - 177 g.
Micronucleus test: Males weighed between 167 g - 206 g.
Animal age on dispatch and on Day 1 of dosing was:
Preliminary toxicity test: On Dispatch Males and females ca 42-48 days old.
Day 1 Males and females ca 47-53 days old.
Micronucleus test: On Dispatch Males ca 42-48 days old.
Day 1 Males ca 47-53 days old.

After arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and given a unique tail mark. Each group was kept with the sexes separated in cages. The animals were kept in a controlled environment with the thermostat and relative humidity within target ranges of 20 to 24°C and 40 to 70% respectively, throughout the study. The room was illuminated by artificial light for 12 hours per day.
All animals were allowed free access to pelleted Envigo Teklad 2014C diet and tap water ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preliminary Toxicity Test concentration: 200 mg/ml
Main test concentration: 50, 100, 200 mg/ml
Duration of treatment / exposure:
The test item was administered on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose, 3 hours before sampling.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 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)
The positive control for the Comet test was Ethyl Methanesulphonate (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

Details of tissue and slide preparation:
Following electrophoresis three slides per animal per tissue were analysed for comets. Slides were visualised by staining with SYBR GOLD® via fluorescence microscopy. 150 morphologically normal cells were analysed for the presence of comets per animal per tissue.
All tissue samples were prepared using identical methods, the cause of the hedgehog cells is not considered to be mechanical/enzyme induced damaged during sample preparation. In addition to this, the potent genotoxin EMS (positive control for the comet phase) caused large increase in the % TI in all tissues analyzed.

Comet Phase
Glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer.
Sections of the liver, glandular stomach and duodenum were placed into ice cold mincing solution, all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method.

For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped.
Comet slides were prepared from all cell suspensions.
Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored at 2 - 8ºC overnight prior to electrophoresis.

Micronucleus Phase
One femur was dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the heads of the femurs removed from each bone. The bone marrow of one femur from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal bovine calf serum by aspiration.
The resulting cell suspensions were centrifuged at 1000 rpm (150 g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal bovine calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).
The slides were fixed in methanol and allowed to air dry. They were then rinsed in purified water and stained using an acridine orange solution at 0.0125 mg/mL and stored at room temperature in the dark until required. Prior to scoring the slides were wet mounted with coverslips using purified water.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Micronucleated polychromatic erythrocytes
Toxicity:
no effects
Remarks:
Animals were treated with Everzol Red CDN Crude at dose levels of 500, 1000 and 2000 mg/kg/day. No clinical signs of toxicity were observed for Everzol Red CDN Crude at any dose level.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Comet slide analysis
Toxicity:
yes
Remarks:
Cytotoxic changes related to treatment with Everzol Red CDN Crude were observed in the stomach (inflammation) and duodenum (degeneration) at 1000 and 2000 mg/kg/day.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose levels of 500, 1000 and 2000 mg/kg/day were selected for the main test.
The main test was carried out in male animals only. No mortalities were observed throughout the duration of the test. Detailed body weights are presented in Appendix 1.
Table 1 gives a summary of the results and statistical analysis of the comet phase. The results for individual animals for the comet phase are presented in Table 2 and hedgehog cell data are presented in Table 3.
Table 4 gives a summary of the results and statistical analysis of the micronucleus phase. The results for individual animals for the micronucleus phase are presented in Table 5.

----Cinical Signs------------
No clinical signs of toxicity were observed for the vehicle control, and positive control or animals administered Everzol Red CDN Crude at any dose level.
Small incidences of body weight loss were observed throughout the main test.

----Tail Intensity------------
The vehicle control group mean % TI values for the liver, duodenum and glandular stomach of Crl:CD(SD) rats were within the 95% confidence limits of the current vehicle historical control range for the individual tissues.
The positive control compound, EMS, produced a statistically significant increase (p<0.001) in the group median % TI when compared to the vehicle control values in all tissues analysed and all % TI values were comparable to the historical positive control range.
There were no statistically significant increases in the group median % TI in the liver of male Crl:CD(SD) rats administered Everzol Red CDN crude at 500, 1000 or 2000 mg/kg/day, compared to vehicle control values. The group mean and median % TI values from all treatment groups were within the 95% confidence limits of the current vehicle historical control range.
There were no statistically significant increases in the group median % TI in the duodenum and glandular stomach of male Crl:CD(SD) rats administered Everzol Red CDN crude at 500 mg/kg/day, compared to vehicle control values. The group mean, and median % TI values were within the 95% confidence limits of the current vehicle historical control range.
There was a statistically significant increase (p<0.001) in the group median % TI observed in the duodenum and glandular stomach of male Crl.CD(SD) rats administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day. The group mean % TI values for animals administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day were within the 95% confidence limits of the current vehicle historical control range.

----Hedgehog Cell------------
There were no hedgehog cells observed in the liver of male Crl:CD(SD) rats administered Everzol Red CDN crude at any dose level.
There was a significant increase in the number of hedgehog cells observed in the duodenum and glandular stomach of male Crl:CD(SD) rats administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day, compared to the concurrent vehicle control

----Micronucleated Polychromatic Erythrocyte Counts (MPCE)------
There were no statistically significant increases in the group %MPCE observed in male Crl:CD(SD) rats administered Everzol Red CDN crude at any dose level, compared to vehicle control values. All group mean values were within the current vehicle historical range (95% confidence limits).

----Micronucleated Normochromatic Erythrocyte (MNCE)------
There were no substantial increases in the incidence of micronucleated normochromatic erythrocytes in male Crl:CD(SD) rats administered Everzol Red CDN Crude at any dose level.

----Proportion of Polychromatic Erythrocytes (%PCE)-------
There were no statistically significant decreases in the group %PCE observed in male Crl:CD(SD) rats administered Everzol Red CDN crude at any dose level, compared to vehicle control values. The group mean %PCE values were all within the current vehicle historical control range (95% confidence limits).

----Histopathology-------
cytotoxic changes related to treatment with Everzol Red CDN Crude were observed in the stomach (inflammation) and duodenum (degeneration) at 1000 and 2000 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
Comet Phase

It is concluded that Everzol Red CDN Crude did not show any evidence of causing an increase in DNA strand breaks in the liver of male Crl:CD(SD) rats administered Everzol Red CDN Crude orally by gavage in this in vivo test procedure.
It is concluded that Everzol Red CDN Crude has shown evidence of causing an increase in DNA strand breaks in the duodenum and glandular stomach of male Crl:CD(SD) rats when administered orally by gavage in this in vivo test procedure. However, these increases in DNA strand breaks are directly associated with an increase in hedgehog cells; although the exact cause of these cellular phenomena is uncertain, it is clear that this would have an impact on the comet response. In addition, histopathology showed evidence of cytotoxic changes related to treatment with Everzol Red CDN Crude which were consistent with the increase in DNA damage seen in the comet assay. Therefore, it is unlikely that the DNA strand breaks observed in the duodenum and glandular stomach are representative of a true genotoxic response.
Micronucleus Phase

It is concluded that Everzol Red CDN Crude has not shown any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male Crl.CD(SD) rats when administered orally by gavage in this in vivo test procedure.
Executive summary:

This study was designed to assess the potential of Everzol Red CDN Crude to induce DNA strand breaks in the liver, glandular stomach and duodenum of Crl: CD(SD) rats and also assess the potential induction of micronuclei by Everzol Red CDN Crude in the bone marrow cells of Crl:CD(SD) rats.

Animals were treated with Everzol Red CDN Crude orally by gavage on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose, 3 hours before sampling.

The vehicle control group received water (purified by reverse osmosis) and the positive control group for the comet phase receivedEthyl methanesulphonate(EMS)at 200 mg/kg,dosed on a single occasion,approximately 3 hours before sampling.

All animals in the vehicle control, test item dose groups and the positive controls were dosed orally by gavage using a dose volume of 10 mL/kg.

On the basis of results from the preliminary toxicity test, dose levels of 500, 1000 and 2000 mg/kg/day were selected for the main test. No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, in line with current guidelines the main test was performed using male animals only.

Comet Phase

Cell suspensions from the liver, glandular stomach and duodenum were obtained fromanimals in the vehicle control group and in each of the test item groups approximately 3 hours after administration of the third dose. Cell suspensions from animals in the positive control group were obtained approximately 3 hours after a single dose.

DNA strand breaks were assessed by comparing the group mean and median % tail intensities (% TI) from Everzol Red CDN Crude treated animals with the concurrent vehicle control values. The slides were also examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells
(i.e. Hedgehog cells).
 These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.

Micronucleus Phase

Bone marrow smears were obtained fromanimals in the vehicle control and in each of the test item groups 3 hours after administration of the third dose.

One smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

Discussion

 

Hedgehogs cells are heavily damaged cells which exhibit a specific morphology (a small or non-existent head and a large diffuse tail); they are considered to be associated with cytotoxicity, necrosis or apoptosis; however, the exact etiology of hedgehog cells is ambiguous. Other evidence suggests they may be caused by mechanical/enzyme induced damage during sample preparation or extreme effects of test chemical genotoxicity (OECD 489, 2016).

 

There were no hedgehog cells observed in the vehicle control animals; asall tissue samples were prepared using identical methods, the cause of the hedgehog cells is not considered to be mechanical/enzyme induced damaged during sample preparation. In addition to this, the potent genotoxin EMS (positive control for the comet phase) caused large increase in the % TI in all tissues analyzed;however, there were no hedgehog cells observed. Therefore, it is unlikely that the increases in hedgehog cells are due to an extreme effect of test item genotoxicity.

 

The hedgehog cells observed in the duodenum and glandular stomach of male animals administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day are considered to be directly related to the increases in the % TI, further confirmed by the plateau in %TI and hedgehog cells in the glandular stomach. Although the exact cause of these cellular phenomena is uncertain, it is clear that they, or their cause, have in this study compromised the comet response.

 

The guidance is clear that in the presence of hedgehog cells or clear cytotoxicity any relevant increases should be interpreted with care. It is considered that the increases obtained in the duodenum and glandular stomach, are unlikely to be of genotoxic origin.

 

Histopathology performed on dose levels 1000 and 2000 mg/kg/day along with vehicle control samples for the glandular stomach and duodenum confirmed inflammation (stomach) and degeneration (duodenum) were consistent with an increase in DNA damage seen in the comet assay.