Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
genetic toxicity in vivo, other
Remarks:
Rat Micronucleus and Alkaline Comet Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phase of the study was performed between 1 June 2015 - 1 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
yes
Remarks:
During Main Experiment 1, the humidity of the animal dosing room went above the Protocol specified range of 45-65% on a single occasion to 66%
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Induction of Micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow. DNA damage in the liver and duodenum of the same animal. Oxidative damage using a hOGG1 modified Comet assay.

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-hex-2-enal
EC Number:
229-778-1
EC Name:
trans-hex-2-enal
Cas Number:
6728-26-3
Molecular formula:
C6H10O
IUPAC Name:
trans-hex-2-enal
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Two consignments were used during the study:
1.used during Experiments 1 and 2
- batch number of test material: 1002362825
- Expiration date of the lot/batch: Received on 13 May 2015 and the best before date was
given as 24 January 2016
- Purity test date: 99.5%

2.used during Experiments 3
- batch number of test material: 0000946637
- Expiration date of the lot/batch: received on 24 November 2016 and the best before date was given as 04 April 2017.
- Purity test date: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, under nitrogen and protected from light

- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Formulations were freshly prepared prior to each dosing occasion by formulating Trans-2-hexenal in corn oil. No stability information was available at the time of animal dosing. Consequently, all formulations were stored at 15-25°C, protected from light, and used within 2 hours of preparation.To ensure homogeneity, dose formulations were stirred continuously (on a magnetic stirrer) immediately before and throughout dosing.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Young adult out-bred Han Wistar rats.
The rat was selected as there is a large volume of background data in both end-points for this species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, UK.
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Exp 1 244 - 306 g; Exp2: 245-301g; Exp3: 263-303g
- Number of animals used: Exp 1: 30 male (includes 3 satellite animals for bioanalysis) ; Exp 2: 30 male (includes 3 satellite animals for bioanalysis) ; Exp 3: 27 male
- Assigned to test groups randomly under following basis: On arrival animals of the same sex were randomly allocated to cages. Range-Finder and satellite animals were allocated to groups of up to three and Main Experiment animals were allocated to groups of six (positive control animals were allocated to groups of three).
Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight of each sex.
- Housing: Animals were housed in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet (e.g. ad libitum): ad libitum , 5LF2 EU Rodent Diet 14% (LabDiet Richmond, Illinois)
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45 to 65%
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark. The animals were routinely kept under these conditions except for short periods of time where experimental procedures dictated otherwise.
- Acclimation: All animals were given a clinical inspection for ill health on arrival. They were acclimatised for at least 5 days and a health inspection was performed before the start of dosing to ensure their suitability for the study.
Fasting: Animals were not fasted prior to administration.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 50 and 35 mg/ml (range-finder); 8.75, 17.5 and 35 mg/ml in main experiments 1,2 and 3
From the results of the Range-Finder Experiment, dose levels of 87.5, 175 and 350 mg/kg/day Trans-2-hexenal (equivalent to 25% MTD, 50% MTD and the MTD respectively) were tested in the Main Experiments.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were freshly prepared prior to each dosing occasion by formulating Trans-2-hexenal in corn oil as outlined: The test article was weighed into a suitable formulation bottle and the appropriate volume of vehicle was added. Formulations were then stirred to mix.




Duration of treatment / exposure:
The test article, vehicle and positive control were given as three administrations, at 0, 24 and 45 hours. All animals were sampled at 48 hours.
All doses were administered at a dose volume of 10 mL/kg. Individual dose volumes were based on individual body weight.
Frequency of treatment:
Three administrations, at 0, 24 and 45 hours
Post exposure period:
An individual record was maintained of the clinical condition of all Range-Finder and Main Experiment animals dosed in the study. Satellite animals were for blood sampling purposes only and were monitored but no individual records of clinical conditions were made. All animals were sampled at 48 hours.
Observation times were as follows:
- Range-Finder Experiment, Day 1,2 and 3, Prior to dose, immediate, 0.5, 1, 2, 4 and 8 hours post dose
- Main Experiments 1, 2 and 3, Day 1and 2, Prior to dose, immediate, 1, 2, 4 and 8 hours post dose
- Main Experiments 1, 2 and 3, Day 3, Prior to dose, immediate and prior to necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
No clinical signs of toxicity
Dose / conc.:
175 mg/kg bw/day (nominal)
Remarks:
No clinical signs of toxicity
Dose / conc.:
87.5 mg/kg bw/day (nominal)
Remarks:
No clinical signs of toxicity
No. of animals per sex per dose:
Six animals per group / males (three in the positive control group).
Main Experiment animals were dosed in replicate cage order: i.e. males from Cage 1 of Groups 1 4, Groups 6-9 or Groups 11-14 dosed in ascending group order then males from Cage 2 of Groups 1-4, Groups 6-9 or Groups 11-14 in ascending group order. Positive control animals (Groups 5, 10 and 15) were dosed on completion of Group 4, 9 or 14 dosing.
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, Ethyl methanesulfonate (EMS, Sigma-Aldrich Chemical Co, Poole, UK), freshly prepared in purified water and administered at 150 mg/kg/day, at 0, 24 and 45 hours.

Examinations

Tissues and cell types examined:
Experiment 1: Bone marrow (Micronucleus assessment), liver and duodenum (Comet analysis).
Experiment 2: liver and duodenum (Comet analysis). Liver was analysed in the Comet assay as this is the primary site of metabolism and is expected to be exposed to both the parent compound and any metabolites. Duodenum was also analysed in the Comet assay as a first site of contact.
Experiment 3: liver (Comet analysis with hOGG1 modification).

Trans-2-hexenal had been tested using a MutaTM Mouse assay with an in vivo peripheral blood micronucleus component included (Beevers, 2013). However, as the final peripheral blood sample was sampled 72 hours after treatment the reliability of the data was considered by the reviewing panel to have been reduced.
Trans-2-hexenal had also been previously tested in a stand-alone micronucleus study in mice and found to be negative (Honarvar, 2007). However, there was no direct confirmation that the bone marrow was exposed as no toxicokinetic measures in plasma were made (EFSA, 2014).
Details of tissue and slide preparation:
Tissues were sampled at necropsy (Day 3, 48 hours).
Histopathology: Preserved liver (Main Experiments 1 and 2) and duodenum (Main Experiment 1) samples were embedded in wax blocks and sectioned at 5 μm nominal. Slides were stained with haematoxylin and eosin and examined by the Study Pathologist.

Bone Marrow Sampling and Micronucleus Slide Preparation: Both femurs were cleaned of adherent tissue and the ends removed from the shanks. Bone marrow was flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose (Sun et al., 1999). Once the majority of the 2 mL had passed through the column a further 4 mL of serum was added to the sample tubes and loaded onto the columns. Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, 15-25°C) and the supernatant aspirated and discarded. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again (as described above) and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of four uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the
labelled slide.

Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. One slide per animal was immediately stained for 5 minutes in 12.5 μg/mL Acridine Orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. Unstained slides were air-dried and initially stored at <-10°C with desiccant. Once final results were confirmed the reserve slides were transferred to storage at room temperature.

Preparation of Comet Cell Suspensions: The Comet liver samples were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 μm) with approximately 4 mL of Merchants solution to produce single cell suspensions.

The Comet duodenum samples were washed thoroughly in Merchants solution and placed into fresh buffer. Each sample was vortexed in Merchants solution for approximately 15 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped (released material discarded) using the back of a scalpel blade. The tissue was vortexed in Merchants solution for a further 15 seconds prior to gently scraping the inside of the duodenum with the back of a scalpel blade. All cell suspensions were held on ice prior to slide preparation.

Preparation of Comet Slides: For Groups 1 to 10 and Group 15 three slides, labelled ‘A’, ‘B’ and ‘C’, were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 30 μL of each single cell suspension was added to 300 μL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 μL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.
For Groups 11-14, six slides were prepared per animal (identified as ‘A’, ‘B’, ‘C’, ‘D’, ‘E’ and ‘F). Slides ‘A’, ‘B’ and ‘C’ were intended for standard Comet/hedgehog analysis, and slides ‘D’, ‘E’ and ‘F’ were for hOGG1 Comet analysis.

Cell Lysis: Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X-100, 10% DMSO) at 2-8°C protected from light. Slides were lysed overnight. At the end of the lysis period slides were processed as follows:
• standard Comet slides A, B and C proceeded immediately to unwinding and
electrophoresis
• hOGG1 Comet Slides D, E and F underwent enzyme treatment (as described
below) prior to unwinding and electrophoresis.

hOGG1 Treatments: Following lysis, slides ‘D’, ‘E’ and ‘F’ for hOGG1 Comet analysis were washed in enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA and 0.2 mg/mL BSA (pH 8.0)) for 2 x 5 minutes at 15-30°C. hOGG1 was added to the gel on each slide in 50 μL enzyme buffer (0.08 U per gel). Coverslips were added to each gel and all slides were incubated for 10 minutes at 37±1°C. Following incubation the coverslips were removed and the slides processed through unwinding and electrophoresis as for the Comet slides (‘A’, ‘B’ and ‘C’).

In-vitro L5178Y Potassium Bromate Treatments: In order to confirm that oxidative damage could be detected in the Comet assay following hOGG1 treatments, in Main Experiment 3 L5178Y cells were treated with potassium bromate, which is known to induce oxidative DNA damage. These in vitro treatments were performed concurrently with the in vivo treatments in accordance with described methods (Smith et al., 2006).
The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Full details of the supplier and mycoplasma testing are documented in central records.
Following treatment, cultures were incubated for 3 hours at 37±1°C. At the end of the incubation, cells were pelleted and resuspended in 300 μL LMA. Triplicate slides per culture (‘A’, ‘B’ and ‘C’ for replicate 1 and ‘D’, ‘E’ and ‘F’ for replicate 2) were prepared as described previously. Once gelled, slides were lysed and processed as follows:
• ‘A’, ‘B’ and ‘C’ slides from replicate 1 cultures were processed as described previously for standard Comet slides
• Slides ‘D’, ‘E’ and ‘F’ from replicate 2 cultures underwent hOGG1 treatments after lysis and prior to unwinding, electrophoresis and neutralisation as described previously.

Unwinding and Electrophoresis: Following lysis (or hOGG1 treatments), slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes (duodenum) or 30 minutes (liver). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (duodenum) or 40 minutes (liver). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.
Prior to scoring, the slides were stained with 100 μL of 2 μg/mL ethidium bromide and coverslipped.
Evaluation criteria:
The experimental unit of exposure for in vivo studies is the animal, and all analysis was based on individual animal response.

Treatment of Micronucleus Data: After completion of microscopic analysis and decoding of the data the following were calculated:
1. %PCE for each animal and the mean for each group. The group mean %PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per number of PCE scored) and %MN PCE for each animal and the group mean %MN PCE (±standard deviation). The numbers of MN PCE in the vehicle control animals were compared with the laboratory's historical control data to determine whether the assay was acceptable.
For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation (Lovell et al., 1989). In the intermediate dose (175 mg/kg/day), intra-group variation in micronuclei frequency was observed, therefore the numbers of MN PCE in each treated group were compared with the numbers in the respective vehicle control groups by use of the Wilcoxon rank sum test (Lehmann, 1975). The tests were interpreted with one-sided risk for increased frequency with increasing dose-response.

Vehicle control data for all tissues in all experiments were comparable with the laboratory’s historical vehicle control data.
The EMS positive control induced a statistically significant increase in MN in bone marrow PCE and a statistically significant increase in %tail intensity in all experiments compared to the concurrent vehicle control.
In Experiment 3 the in vitro positive control group (potassium bromate) induced marked, dose related increases in %tail intensity (over the concurrent vehicle control group at 130 and 250 μmol/L) thus demonstrating that oxidative damage can be detected in the Comet assay following hOGG1 treatment. The assay was therefore accepted as valid.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989) and Lehmann E L (1975). Nonparametrics: Statistical methods based on ranks, Ch1.

Levene’s test for equality of variances between the groups was performed to assess inter-group heterogeneity. Where this test showed evidence of heterogeneity (P≤0.01), a rank-transformation was applied to the data prior to analysis.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In all experiments, no clinical signs of toxicity were observed in any animal following treatments with vehicle, Trans-2-hexenal (at 87.5, 175 or 350 mg/kg/day) or the positive control (EMS).
Although no clinical signs of toxicity were observed in the Main Experiments, assessment of a higher dose level (500 mg/kg/day) in the Range-Finder Experiment demonstrated mortality in a single animal. Therefore, 350 mg/kg/day was considered to be an appropriate MTD.

Body Weights: There were dose related decreases in bodyweight change from Days 1 to 3 during Main Experiment 1 (+2.5%, +0.1% and -1.5% at 87.5, 175 and 350 mg/kg/day respectively compared to +3.3% in the vehicle control group).
In Main Experiment 2, there was a decrease in bodyweight change from Days 1 to 3 at 350 mg/kg/day only (-1.4% compared to +0.9% in the vehicle control group). There was no notable effect on bodyweight in any Trans-2-hexenal treated animals in
Main Experiment 3. However, there was a marked decrease in bodyweight change observed in the positive control animals (-9.8% compared to +1.2% in the vehicle control group).

Clinical chemistry: In Main Experiment 1, samples from Animals 1 to 3, 5 to 13 and 24 were found to be lipaemic. Additionally, samples from Animals 5, 12 and 24 were found to be haemolysed.
In Main Experiment 2, samples from Animals 31 to 41, 43, 45, 48 and 50 to 53 were found to be lipaemic. Additionally, samples from Animals 31, 32, 33, 34, 36, 37, 43 and 51 were found to be haemolysed. As this can interfere with analysis, all data were reviewed with caution. The lipaemia was attributed to the final administration of corn oil (either as the vehicle control or as part of the test article formulation) given only 3 hours prior to blood sampling. There were no clinical chemistry findings considered to be related to administration of Trans-2-hexenal.

Histopathology: There were no macroscopic or microscopic findings considered to be related to administration of Trans-2-hexenal.

Micronucleus Analysis: Animals treated with Trans-2-hexenal at 87.5 and 175 mg/kg/day exhibited group mean %PCE values that were similar to the concurrent vehicle control group and which were within the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity. There was a slight decrease in %PCE ratio at 350 mg/kg/day.
However, the decrease was not marked enough to definitively infer bone marrow
toxicity. Trans-2-hexenal treated rats displayed MN PCE frequencies that were generally similar to the concurrent vehicle control group (Text Table 2) and which were consistent with the laboratory's historical data (Section 7.3). Individual frequencies of MN PCE for all treated animals were consistent with historical vehicle control distribution data and similar to frequencies observed in the concurrent controls. There were no statistically significant (Wilcoxon rank sum test) increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control group.
These data confirm that Trans-2-hexenal did not induce MN in the bone marrow of treated male rats (see attachment summary of micronucleus data)

Comet Analysis: For all experiments, there were no marked increases in %hedgehogs in either liver or duodenum cells following treatment with Trans-2-hexenal, thus demonstrating that treatment did not cause excessive DNA damage that could have interfered with Comet analysis

Liver Analysis – Experiment 1
Group mean %tail intensity and tail moment values for all groups treated with Trans-2-hexenal exceeded the group mean vehicle control data but no increases were statistically significant, there was no evidence of a dose relationship and all animals fell within the laboratory’s historical control data. In each group, there were one or two animals displaying elevated %tail intensity and tail moment values which increased the group mean (87.5 mg/kg/day, Animal 10; 175 mg/kg/day, Animals 13 and 14 and 350 mg/kg/day, Animal 22 (Table 9.7)). With the exception of these animals, all individual animal data at all dose levels were generally comparable with the vehicle control animals. Due to the spurious nature of some of the individual animal responses, it was decided following consultation with the Sponsor, that the Experiment be repeated in order to obtain further data.

Liver Analysis – Experiment 2
Group mean %tail intensity and tail moment values for all groups treated with Trans-2-hexenal exceeded the group mean vehicle control data (Text Table 4). At the high dose (350 mg/kg/day), these increases were found to be statistically significant (p≤0.05) and there was also a statistically significant linear trend (p≤0.01). Individual animal %tail intensities in the 87.5 mg/kg/day dose group were in the range of 0.21-1.46, the 175 mg/kg/day dose group were in the range of 0.43-1.16 and the 350 mg/kg/day dose group were in the range of 0.25-2.68 (Table 10.6) compared to the vehicle control range of 0.08-1.03 (the 95% reference range of the historical control data was 0.04-5.50). At least three animals in each test article treated group were found to overlap with the concurrent vehicle control and all animals fell within the laboratory’s historical control data. Following further consultation with the Sponsor, it was decided that further data were required. A modified Comet assay was conducted which examined the potential for Trans-2-hexenal to induce oxidative DNA damage.

Liver Analysis – Experiment 3
In Experiment 3 without hOGG1 modification animals treated with Trans-2-hexenal at all doses exhibited %tail intensities and tail moments that were similar to the concurrent vehicle control group (Text Table 5 and Table 11.5) and that fell within the laboratory's historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control. In Experiment 3 with hOGG1 modification animals treated with Trans-2-hexenal at all doses exhibited %tail intensities and tail moments that were similar to the concurrent vehicle control group. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control confirming that Trans-2-hexenal did not induce oxidative DNA damage (see attachment: Summary of Experiment 3 Group Mean Comet Data - Liver)
Taking into account the data from all experiments, the data do not fulfil the criteria for a positive result as per OECD 489 guideline. Small increases in %tail intensity were observed in sporadic animals over two separate experiments in the liver, but only achieved statistical significance at one dose in one of these experiments. In a third experiment no increases in %tail intensity were observed and hOGG1 modification did not reveal any evidence of oxidative DNA damage. Therefore, the isolated increases were not readily reproducible and were considered to be biologically irrelevant.

Duodenum Analysis – Experiment 1
Group mean %tail intensity and tail moment values for all groups of animals treated with Trans-2 hexenal were smaller than the group mean vehicle control data. There were no statistically significant increases in mean %tail intensity between treated groups and the vehicle control group. All individual animal data at all dose levels were generally consistent with the vehicle control animals and fell within the laboratory’s historical control data. The only exceptions to this were Animal 14 (175 mg/kg/day) and Animals 21 and 23 (350 mg/kg/day) which fell below the laboratory’s historical control 95% reference range. However, mean %tail intensity values for all test article treated groups were within the laboratory’s historical vehicle ranges.

Duodenum Analysis – Experiment 2
Group mean %tail intensity and tail moment values for all groups of animals treated with Trans-2-hexenal were comparable with the group mean vehicle control data. There were no statistically significant differences in %tail intensity between treated groups and the vehicle control group. All individual animal data at all dose levels were generally consistent with the vehicle control animals
and fell within the laboratory’s historical control data (Section 7.4). The only
exceptions to this were Animals 37, 40 and 42 (87.5 mg/kg/day) and Animal 54
(350 mg/kg/day) which fell below the laboratory’s historical control 95% reference
range. However, mean %tail intensity values for all test article treated groups were within the laboratory’s historical vehicle ranges.
These data confirm that Trans-2-hexenal did not induce DNA damage in the duodenum of treated male rats.

Any other information on results incl. tables

See attachements

Applicant's summary and conclusion

Conclusions:
It is concluded that under the conditions of this study, Trans-2-hexenal, did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow in male rats treated up to 350 mg/kg/day (an estimate of the maximum tolerated dose for this study). In the same animals, Trans-2-hexenal did not induce DNA damage in the duodenum or liver, as analysed by the Comet assay. In the liver small increases in %tail intensity were observed in sporadic animals over two separate experiments, but only achieved statistical significance at one dose in one of these experiments. In a third experiment no increases in %tail intensity were observed and hOGG1 modification did not reveal any evidence of oxidative DNA damage. Therefore, the isolated increases were not readily reproducible and were considered to be biologically irrelevant.
Executive summary:

Trans-2-hexenal was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats and to induce DNA damage in the liver and duodenum of the same animals. Further data were generated in two additional experiments including examining the potential for
inducing oxidative damage using a hOGG1 modified Comet assay.

This study was designed to provide data from two genetic toxicology end-points whilst minimising the number of animals used.

Micronucleus Data Summary
Animals treated with Trans-2-hexenal at 87.5 and 175 mg/kg/day exhibited group mean %PCE values that were similar to the concurrent vehicle control group and which were within the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity. There was a slight decrease in %PCE ratio at 350 mg/kg/day. However, the decrease was not marked enough to definitively infer bone marrow toxicity.
Trans-2-hexenal treated rats displayed MN PCE frequencies that were generally similar to the concurrent vehicle control group and which were consistent with the laboratory's historical data. Individual frequencies of MN PCE for all treated animals were consistent with historical vehicle control distribution data and similar to frequencies observed in the concurrent controls. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control group.


Comet Data Summary - Liver
For all experiments there were no marked increases in %hedgehogs following treatment with Trans-2-hexenal, thus demonstrating that treatment did not cause excessive DNA damage that could have interfered with Comet analysis. Experiment 1, group mean %tail intensity and tail moment values for all groups treated with Trans-2-hexenal exceeded the group mean vehicle control data but no increases were statistically significant, there was no evidence of a dose relationship and all animals fell within the laboratory’s historical control data. In each group, there were one or two animals displaying elevated %tail intensity and tail moment values which increased the group mean (87.5 mg/kg/day, Animal 10; 175 mg/kg/day, Animals 13 and 14 and 350 mg/kg/day, Animal 22). With the exception of these animals, all individual animal data at all dose levels were generally comparable with the vehicle control animals.

In Experiment 2, group mean %tail intensity and tail moment values for all groups treated with Trans-2-hexenal exceeded the group mean vehicle control data. At the high dose (350 mg/kg/day), these increases were found to be statistically significant and there was also a statistically significant linear trend. Individual animal %tail intensities in the 87.5 mg/kg/day dose group were in the range of 0.21-1.46, the 175 mg/kg/day dose group were in the range of 0.43-1.16 and the 350 mg/kg/day dose group were in the range of 0.25-2.68 compared to the vehicle control range of 0.08-1.03 (the 95% reference range of the historical control data was 0.04-5.50). At least three animals in each test article treated group were found to overlap with the concurrent vehicle control and all animals fell within the laboratory’s historical control data.

In Experiment 3 without hOGG1 modification animals treated with Trans-2-hexenal at all doses exhibited %tail intensities and tail moments that were similar to the concurrent vehicle control group and that fell within the laboratory's historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control.

In Experiment 3 with hOGG1 modification animals treated with Trans-2-hexenal at all doses exhibited %tail intensities and tail moments that were similar to the concurrent vehicle control group. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control confirming that Trans-2-hexenal did not induce oxidative DNA damage. Taking into account the data from all experiments, the data do not fulfil the criteria for a positive result as per OECD 489 guideline. Small increases in %tail intensity were observed in sporadic animals over two separate experiments in the liver, but only achieved statistical significance at one dose in one of these experiments. In a third experiment no increases in %tail intensity were observed and hOGG1 modification did not reveal any evidence of oxidative DNA damage. Therefore, the isolated increases were not readily reproducible and were considered to be biologically irrelevant.

Comet Data Summary - Duodenum
For both Experiments 1 and 2, there were no dose related increases in %hedgehogs following treatment with Trans-2-hexenal, thus demonstrating that treatment did not cause excessive DNA damage that could have interfered with Comet analysis. Following Main Experiment 1, group mean %tail intensity and tail moment values for all groups of animals treated with Trans-2-hexenal were smaller than the group mean vehicle control data. There were no statistically significant increases in mean %tail intensity between treated groups and the vehicle control group. All individual animal data at all dose levels were generally consistent with the vehicle control animals and fell within the laboratory’s historical control data. The only exceptions to this were Animal 14 (175 mg/kg/day) and Animals 21 and 23 (350 mg/kg/day) which fell below the laboratory’s historical control 95% reference range. However, mean %tail intensity values for all test article treated groups were within the laboratory’s historical vehicle ranges. Following Main Experiment 2, group mean %tail intensity and tail moment values for all groups of animals treated with Trans-2-hexenal were comparable with the group mean vehicle control data. There were no statistically significant differences in %tail intensity between treated groups and the vehicle control group. All individual animal data at all dose levels were generally consistent with the vehicle control animals and fell within the laboratory’s historical control data. The only exceptions to this were Animals 37, 40 and 42 (87.5 mg/kg/day) and Animal 54 (350 mg/kg/day) which fell below the laboratory’s historical control 95% reference range. However, mean %tail intensity values for all test article treated groups were within the laboratory’s historical vehicle ranges.

Conclusion

It is concluded that under the conditions of this study, Trans-2-hexenal, did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow in male rats treated up to 350 mg/kg/day (an estimate of the maximum tolerated dose for this study). In the same animals, Trans-2-hexenal did not induce DNA damage in the duodenum or liver, as analysed by the Comet assay. In the liver small increases in %tail intensity were observed in sporadic animals over two separate experiments, but only achieved statistical significance at one dose in one of these experiments. In a third experiment no increases in %tail intensity were observed and hOGG1 modification did not reveal any evidence of oxidative DNA damage. Therefore, the isolated increases were not readily reproducible and were considered to be biologically irrelevant