Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Comet assay / OECD 489
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 JUN 2020 to 4 NOV 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted 29 JUL 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexanal
EC Number:
200-624-5
EC Name:
Hexanal
Cas Number:
66-25-1
Molecular formula:
C6H12O
IUPAC Name:
hexanal
additive 1
Chemical structure
Reference substance name:
3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-benzopyran-6-ol
EC Number:
233-466-0
EC Name:
3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-benzopyran-6-ol
Cas Number:
10191-41-0
Molecular formula:
C29H50O2
IUPAC Name:
2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-ol
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rats are routinely tested in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at the lab.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop ZRT. H-1103 Budapest, Cserkesz u. 90.
- Age at study initiation: 50 - 52 d
- Weight at study initiation: 222 - 246 g
- Assigned to test groups randomly: No. All animals were sorted according to body weight by computer and grouped according to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping will be controlled by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.
- Housing: 3 animals / cage; at the positive control group 2 animals / cages in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm - width x length x height) cages on certified laboratory wood bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 22.9
- Humidity (%): 41 - 66
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sunflower oil
- Justification for choice of solvent/vehicle: The applicability of test item formulations in sunflower oil was additionally checked in the frame of the present study in a non-GLP preliminary solubility test. The testing laboratory has validated an analytical method for the necessary dose formulation analysis and additionally an in-study partial validation was performed where recovery and stability of the 450 mg/mL test item solution was investigated. At the chosen target tissues, the suitability of sunflower oil vehicle in the in vivo Comet assay was evaluated in separate validation, reliability studies performed in the testing laboratory under the same conditions as the present study; furthermore it is confirmed with an available own laboratory’s historical control database: that is based on four experiments in the case of stomach and liver, and on two experiments in the case of kidney.
- Concentration of test material in vehicle: 400, 200 and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 5 mL/kg bw (test item), 10 mL/kg bw (positive control)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Formulations were prepared before each treatment.
Duration of treatment / exposure:
24h
Frequency of treatment:
twice, once on the day 0 and 24 hours thereafter
Post exposure period:
3-4 hours after the second treatment (doses and vehicle control)
3-4 hours after the treatment (positive control)
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
6 / dose group and negative control group, 4 / positive control group
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (EMS)
- Justification for choice of positive control(s): Positive control group with known mutagen
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw

Examinations

Tissues and cell types examined:
- Target tissues for sample preparation: Stomach, liver, kidney and testis seminiferous tubules (gonadal cells)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of previous studies the maximum dose was set to 2000 mg/kg bw/day (the maximum limit dose). Furthermore this dose is the maximum dose recommended in the respective OECD guideline. Two additional doses, 1000 and 500 mg/kg bw/day were selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treatment: twice; once ontrypan day 0 and 24 hours thereafter
Sampling times: 3-4 hours after the second treatment

DETAILS OF SLIDE PREPARATION:
- After pre-treatment of the slides the cells were embedded. Before the use a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA (see above) and covered with a glass coverslip. The slides were placed on a tray until the agarose hardens (~ 5 minutes). After the cell isolations each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA). Thereafter 85 or 165 μL (~1-9 x 104 cells) of this mixture was added on the microscope slide after gentle slide off the coverslip*. The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardens an additional 70 μL of NMA was dropped on the microscope slide after a gentle slide off the (second) coverslip and an additional new coverslip was laid on the slide. After the repeated NMA layer hardens the coverslip was removed.

METHOD OF ANALYSIS:
- Cytotoxicity was determined (as a first screening) on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling.
- DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis).
- For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment and per vehicle control; 450 per positive control).
Evaluation criteria:
test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
Statistics:
The statistical significance of % tail DNA values, tail length and OTM values; furthermore, the number of ghost cells was carried out using the appropriate statistical method, using SPSS software. The homogeneity of variance between groups was checked by Bartlett's homogeneity of variance test, the normal distribution of data was examined by Kolmogorov-Smirnov test. Following these analyses, a one-way analysis of variance following by Dunnett’s test was used to compare the vehicle control value and the corresponding values of test item doses and positive control.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
no statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, liver or kidney cells
Toxicity:
no effects
Remarks:
No cytotoxicity (using Trypan blue dye exclusion method) was noticed in any test and control item treatments for any target tissue. Number of ghost cells in respective tissues did not differ from vehicle control or were within historical control data.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Based on available information of previous studies the performance of a range finding study was not necessary. The highest dose was selected according to the criteria required by the OECD 489 Guideline.

Any other information on results incl. tables

Results

- All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met.

- At the highest concentration no mortality was observed.

- Expect eating of embedding of 2000 mg/kg bw/day, no toxic symptoms or any clinical signs were observed during the treatments in the remaining dose levels and controls.

- At the tissue isolation normal appearance and anatomy of liver, kidney and testis was noticed in all dose levels and controls.

- At the stomach openings test item smell and characteristic stomach content (bedding material or at 1000 mg/kg body weight/day in two cases conspicuously thin liquid) was noticed. Additionally, in several cases stomach tympanites were noticed. Smell intensity and bedding amount in the stomach showed a dose dependent tendency.

- No effect on body weights were stated.

- No cytotoxicity was noticed in any test and control item treatments for any target tissue (using Trypan blue dye exclusion method) .

- Number of ghost cells in the liver and kidney samples remained nearly in the same range and did not differ statistically significantly from that of the vehicle control.

- Despite slight dose related tendency and statistical difference from vehicle control (at highest dose level), number of ghosts cells in the stomach remained well within historical control data. The relatively higher number of ghost cells in stomach samples especially at the highest dose level seemed to be associated with toxic effects attributable to the test item, which was observed during macroscopic inspection of the tissue prior to cell isolation, whereas no cytotoxic effects (see above) were noted. Nevertheless, even at this local toxic effect concentration at the tissue of first contact no increased DNA migration was noted.

- Statistically significant increase of ghost cells was noticed at the EMS treatments in the stomach, liver and kidney samples (in line with historical control data).

- The mean median % tail DNA values of all dose groups did not differ statistically significantly from that of the vehicle control. All mean median % tail DNA values fell within corresponding historical control data.

- The tail length values of the stomach samples did not differ statistically significantly from that of the vehicle control in whole examined dose range. Statistically differences were observed in case of samples of the liver (at the two highest doses) and the kidney (at highest dose). However, these significances were considered as not relevant for mutagenicity assessment since the significances were observed for lower values (compared to the concurrent control) noted for the above dose levels. In addition, all values for test and control items were well within the corresponding historical control data ranges

- The Olive Tail Moment values in the stomach, liver and kidney of the test item treated groups did not differ statistically significant from that of the vehicle control and were within the established historical control data ranges.

Applicant's summary and conclusion

Conclusions:
The investigated test item Hexanal 0,05% Toco did not show genotoxic activity in the examined tissues in this Comet Assay.
Executive summary:

The study was performed according to OECD TG 489, is fully reliable and in concordance with GLP.

In the study the test item Hexanal 0,05% Toco was investigated by the means of the in vivo comet assay on isolated stomach, liver and kidney cells under alkaline conditions in the male HAN:WIST rats. The test item was administered twice via oral gavage at the dose levels 2000, 1000 and 500 mg/kg body weight/day. Sampling was performed about 3 to 4 hours after the second treatment. As negative (vehicle) control sunflower oil and as positive control ethyl methanesulfonate (EMS) was used.

Under the experimental conditions presented in this report, the test item Hexanal 0,05% Toco did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, liver or kidney cells. Concurrent controls confirmed the sensitivity and validity of the test.

The investigated test item Hexanal 0,05% Toco did not show genotoxic activity in the examined tissues in this Comet Assay.