Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29. July 2016
tissues: liver, glandular stomach and duodenum
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
EC Number:
248-227-6
EC Name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
Cas Number:
27107-89-7
Molecular formula:
C38H74O6S3Sn
IUPAC Name:
2-ethylhexyl 10-ethyl-4-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecan-1-oate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is one of the recommended species by regulatory agencies for conducting in vivo Comet assay among rodents.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source of supply : In-house bred animals
No. of groups : 5 groups (1 Vehicle Control,1 Positive Control and 3 Treatment groups)
No. of animals /group and sex: 10 (5 Males + 5 Females)
Females used were nulliparous and non pregnant
Age at treatment : 7 weeks
Body weight range at receipt (g): Male - 194.01 to 220.12 g, Female - 190.11 to 217.01 g
Animal identification : Acclimatization Period - Cage cards with minimum details of study no., animal no., sex and tail marking with marker pen.
Treatment Period - All the animals were identified by body marking using turmeric solution, potassium permanganate (f roup five) and additionally, a cage card was displayed which includes study no., cage no., sex, animal no. (permanen reatment date and date of necropsy.

Environmental Conditions: Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate
fresh air supply (12 to 15 air changes per hour), room temperature 19.8 to 22.9°C and relative humidity 48 to 67% with 12 hours light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.

Housing Maximum of three animals of same sex and group were housed together in a standard polypropylene cage
(L 430 × B 285 × H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized paddy husk was used as a bedding material.

Feed: Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to
the animals throughout the experimental period. The contaminant analysis test reports of feed are included as Annexure 4.

Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through rever se osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Peanut Oil
Lot No.: AG07110
Expiry Date: 17/02/2022
Make: MP Biomedicals
Details on exposure:
The test item was administered by the oral route once a day for 2 consecutive days using gavage cannula. The test item was administered in a dose volume of 5 mL/kg with the doses of 100, 200 and 300 mg/kg designated as low (G2), mid (G3) and high dose (G4), respectively. The vehicle control group (G1) animals were administered the peanut oil (vehicle) only at a dose volume of 5 mL/kg body weight.

The positive control group (G5) animals were administered with Ethyl methanesulfonate dissolved in distilled water at the volume of 10 mL/kg body weight. Ethyl methanesulfonate was administered at a dose of 250 mg/kg.

The dose volume to be administered was calculated for individual animals based on treatment day body weight of the animal.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
EMS 250 mg/kg

Examinations

Tissues and cell types examined:
Animals were euthanized by cervical dislocation post 2 to 6 hours after the second treatment. Selected tissue (liver, glandular stomach and duodenum) were removed, dissected and a portion was collected for the comet assay. The tissues for the comet assay were placed into ice-cold mincing and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and were stored in ice- cold mincing buffer until processed. All the animals were subjected to gross pathological examination

Single cell suspensions were prepared by chopping the tissue with scalpels in a small amount of ice-cold mincing solution. The scraped tissue solution was transferred into a homogenization tube and then gently homogenized using a homogenizer. Homogenate was filtered through a 30 micron pore nylon mesh and was then centrifuged at 800xg for 10 minutes. Single cells were suspended in DPBS. Cell suspensions were maintained at 2 to 8°C
Details of tissue and slide preparation:
Three slides were prepared for each tissue from each animal and labelled with the study number, animal no., tissue, slide number (e.g. 1/3 to 3/3) and sex of each animal using pencils. To reduce the possibility of detachment of the agarose during the procedure, slides were pre coated with 100 µL of liquid agarose and the agarose was allowed to dry to a thin film. Approximately 75 µL of cell suspension with 75 µL of
1.0% low-melting agarose gel was mixed and rapidly pipette onto the surface of the pre coated slides and a coverslip was placed on it. Slides were placed on ice packs until the Agarose layer hardened (20 minutes). Slides were immersed in chilled lysing solution in the dark for overnight. After completion of lysing, the slides were rinsed in distilled water to remove residual detergent and salts prior to alkali unwinding step.

Evaluation criteria:
All the slides were coded before evaluation to avoid group bias during evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes and stain with Ethidium bromide, covered with a fresh coverslip and cells were scored under 200 X magnification. At least 150 cells were analyzed per sample. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs were determined of at least 150 cells per sample.
Statistics:
Body weight were analyzed by SPSS at a 95% level of confidence (p<0.05) of significance. Inter group comparison of Body weight (day 1 and day 2) and percent tail DNA. The dose correlation was done using ‘t’ test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
The positive control [G5], Ethyl methanesulfonate at a dose of 250 mg/kg produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (Liver, glandular stomach and duodenum)

Applicant's summary and conclusion

Conclusions:
The data obtained under the conditions employed during this experiment support the conclusion that the test item, MOTE, did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 300 mg/kg.
Executive summary:

The results for the assessment of the test item, MOTE, to cause DNA strand breaks are provided for the doses of 100 [G2], 200 [G3] and 300 [G4] mg/kg, respectively, in male and female Wistar rats. The average % tail DNA observed in liver is 2.43, 2.40, 2.53 and 2.83 in males and 2.54, 2.46, 2.51 and 2.90 in females dosed at 0, 100, 200 and 300mg/kg, respectively. In glandular stomach, the observed average % tail DNA is 3.21, 3.35, 3.75 and3.85 in males and 3.34, 3.41, 3.88 and 3.94 in females dosed at 0, 100, 200 and 300 mg/kg, respectively. The average % tail DNA observed in duodenum was 2.85, 2.88, 2.89 and 3.26 in males and 2.96, 3.07, 3.00 and 3.19 in females dosed at 0, 100, 200 and 300 mg/kg, respectively. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any organ for any of the MOTE groups when compared to the vehicle control group.

The positive control [G5], Ethyl methane sulfonate at a dose of 250 mg/kg produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (Liver, glandular stomach and duodenum) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the COMET assay for this test of MOTE were fully adequate.