Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Comet Assay
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This in vivo study was performed as part of the OECD 422 study. This has been done as based on the positive outcome of the Ames test, an additional in vivo study is warranted. In order to minimize the number of animals necessary for the assessment and to further investigate the mutagenicity endpoint the assessment was incorporated in the repeated dose study as is also described in the guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Principles of method if other than guideline:
Six males from each dose group will be dosed to Day 45 and six N-Nitroso-N-methylurea dosed controls will kept without dosing during day 0-43 and will be dosed from day 43 to 45. For the males from Groups 1 to 4 of the OECD 422 study as well as all positive control males, samples of the liver (after recording of liver weight), glandular stomach and jejunum were processed to provide single cell suspensions with sufficient numbers of cells for the Comet Assay. The procedure was performed under subdued lighting and the Comet assay tissues/cells were processed as quickly as possible, using ice-cold buffers to maintain the tissues and cell preparations at low temperature.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
dentification: C.I. Direct Yellow 133
Physical state/Appearance: Brown/orange solid flakes
Expiry Date: 01 September 2021
Storage Conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Strain: Wistar Han™:RccHan™:WIST
- Age at study initiation: 11 weeks
- Weight at study initiation: 274-361 g
- Housing: group housed up to three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK) (except during the mating period of the OECD 422 study)
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ad libitum
- Water: ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at lest 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil for treatment groups (4 mL/kg), distilled water for positive control (N-Nitroso-N-methylurea ,10 mL/kg)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
the test item was prepared at the appropriate concentrations as a suspension in Corn oil. Formulations were made daily from Days 1 to 8 of dosing, and dosed within four hours of being prepared. Following confirmed stability and homogeneity results, formulations were prepared weekly and stored refrigerated in the dark.
N-Nitroso-N-methylurea was dissolved in distilled water at appropriate concentration. A purity adjustment (for 90% purity) was made when preparing dosing formulations. Fresh formulations were prepared each day and dosed within two hours of preparation.

Duration of treatment / exposure:
45 days for treatment groups, 3 days for positive control (undosed during the first 43 days)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
initially dosed at 200 mg/kg bw during day 1-22, reduced to 125 mg/kg bw thereafter (due to mortality and sever weight loss)
Dose / conc.:
2.5 mg/kg bw/day (actual dose received)
Remarks:
N-Nitroso-N-methylurea (positive control)
No. of animals per sex per dose:
6 males for treatment groups, 5 males for positive controls
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
N-Nitroso-N-methylurea
- Justification for choice of positive control(s): according to the guideline and historical control data are available in the laboratory
- Route of administration: gavage
- Doses / concentrations: 2.5 mg/kg bw

Examinations

Tissues and cell types examined:
glandular stomach, jejunum and liver
Details of tissue and slide preparation:
Liver - A small piece of liver was excised (approximately 1 cm2) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.
Glandular Stomach and Jejunum - The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension
was filtered through gauze prior to use for the comet slides.
A section of jejunum (approximately 2 cm2) was removed, cleaned and then immersed in stomach buffer for approximately 15 minutes on ice. A cell suspension was obtained by scraping the tissue of the jejunum into stomach buffer and filtering it through gauze.

Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the Comet assay.
Once the cell suspensions were obtained, approximately 30 μL of the cell suspension was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on
each slide, and 4 gels were prepared for each tissue. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
After the lysis phase had been completed the slides were removed from the lysing buffer, rinsed with neutralization buffer (0.4M Tris pH 7.5) to remove residual detergents and salts and then placed randomly into an electrophoresis unit. The electrophoresis unit was filled with chilled electrophoresis buffer (pH >13) until the slide surface was just covered. The slides were left for approximately 20 minutes to allow the DNA to unwind, after which electrophoresis proceeded at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period the bath was switched off, the slides gently removed and rinsed three times with neutralization buffer for approximately 5 minutes each time. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry, after which they were stored prior to staining and scoring. For each tissue, two processed slide gels were scored blindly. To each dry slide gel, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA, the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PCbased image analysis program (Comet IV version 4.3.1). Two slide gels for each tissue for each animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per ani.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
a) at least one of the test doses exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is considered to be dose-related
c) the results are substantially outside the distribution of the historical negative control range
The test chemical is when all three conditions are met considered to be able to induce DNA strand breakage in the tissues studied

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no evidene of a dose-related response
c) all results are within historical negative control range
d) direct or indirect evidence to demonstrate exposure of, or toxicity to, the target tissue(s) has been achieved
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
liver degeneration, necrosis and apoptosis, jejunum yellow discolouration; stomach green/yellow coloured contents
Vehicle controls validity:
valid
Remarks:
within historical control ranges
Positive controls validity:
valid
Remarks:
within or slightly above historical control values
Additional information on results:
At 100 or 200/125 mg/kg bw/day there were small but statistically significant dose related increases (p<0.05 - p<0.01) in the mean and median percentage tail intensities over the vehicle control value for the liver. However, these increases were all within the laboratory historical control range for the vehicle used, and were set against a relatively low vehicle control value and were therefore considered to be due to cytotoxicity rather than DNA strand breakage. This is confirmed by the results of the histopathology on the liver where degeneration, necrosis and apoptosis were observed. The treatment with the test item was therefore considered not to induce any DNA damage in the liver.

Any other information on results incl. tables

Glandular Stomach

Dose level (mg/kg bw)

Group mean % hedgehogs

Group mean % tail intensity

Group mean of median % tail intensity per animal

Group mean SD % tail intensity

0

4.27

2.35

0.58

4.18

30

4.74

2.08

0.49

3.66

100

5.83

2.72

0.82

4.21

200/125

4.42

2.25

0.63

3.70

25 (MNU)

9.61

34.01

32.32

13.71

 

Jejunum

Dose level (mg/kg bw)

Group mean % hedgehogs

Group mean % tail intensity

Group mean of median % tail intensity per animal

Group mean SD % tail intensity

0

8.72

3.48

1.07

6.06

30

11.05

4.03

1.48

6.06

100

9.25

3.63

1.18

6.01

200/125

10.74

4.00

1.32

6.79

25 (MNU)

16.59

47.53

48.22

15.44

 

Liver

Dose level (mg/kg bw)

Group mean % hedgehogs

Group mean % tail intensity

Group mean of median % tail intensity per animal

Group mean SD % tail intensity

0

0

0.35

0.02

0.85

30

0

0.40

0.03

0.97

100

0

0.52*

0.03*

1.45

200/125

0

0.54**

0.04**

1.27

25 (MNU)

15.33

43.05

43.07

9.09

*P<0.05; ** p<0.01

Historical controls Liver (based on 15-16 experiments)

Dose level (mg/kg bw)

Group mean % hedgehogs

Group mean % tail intensity

Group mean of median % tail intensity per animal

SD % of tail intensity

vehicle

0.00-1.16

0.17-0.95

0.00-0.08

0.55-5.86

Positive control

0.29-10.24

13.65-46.47

13.49-47.10

5.15-13.31

 

Applicant's summary and conclusion

Conclusions:
The substance is considered not to induce toxicologically significant increases in the mean or median percentage tail intensity values in the jejunum, glandular stomach and liver and therefore the substance was considered to be non-genotoxic to these tissues.  
Executive summary:

A Comet assay was performed on male rats that were dosed by gavage for 45 days in an OECD 422 study at 0, 30, 100 and 200/125 mg/kg bw. Rats that remained untreated until day 43 and were treated thereafter until day 45 with 2.5 mg/kg bw N-Nitroso-N-methylurea.

The Comet assay assessment revealed there was a small but statistically significant dose related increases in the percentage tail intensity and median percentage tail intensity of the liver but these were all within the background control ranges for the vehicle control.  The increases were considered to be due to cytotoxicity rather than DNA strand breakage. This was confirmed by the results of the histopathology on the liver where degeneration, necrosis and apoptosis were observed. Therefore the substance was considered not to induce any DNA damage in the liver.  It was concluded that there were no toxicologically significant increases in the mean or median percentage tail intensity values in the jejunum, glandular stomach and liver and therefore the substance was considered to be non-genotoxic to these tissues.