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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
28-day toxicity study
Type of information:
experimental study
Adequacy of study:
other information
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no

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
- Source and lot/batch No.of test material: Hexenal (CAS 6728-26-3) was ontained from Sigma (St. Louis, Missouri).
- Expiration date of the lot/batch: Not reported
- Purity test date: The reported purity of the test item was 98% (test date not reported).


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: The stability of dosing solutions for 1 week was confirmed by NMR spectroscopy before the initiation of the 4-week study; dosing solutions were prepared once weekly.
- Solubility and stability of the test substance in the solvent/vehicle: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Details of the treatment of the test material proir to testing are not reported.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina)
- Age at study initiation: 6 weeks of age
- Weight at study initiation: Not reported
- Fasting period before study: The rats were not fasted before dosing
- Housing: 5 rats/ cage
- Diet: Rat chow (Purina) ad libitium
- Water: ad libitium
- Acclimation period: 1.5 to 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour day/night cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:

- VEHICLE
- Justification for use and choice of vehicle: Not reported. Assumed to be due to the solubility of the test substance to form a suitable formulation for gavage dosing.
- Amount of vehicle (if gavage): Dosing volume 2 ml/kg
- Purity: Not reported
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No details reported.
Duration of treatment / exposure:
Rats were dosed by oral gavage for 5 days per week for 4 weeks, and then killed 1 day after the final dose.
Frequency of treatment:
Rats were dosed once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per dose
Details on study design:
- Dose selection rationale: Details not reported.
- Rationale for animal assignment (if not random): Details not reported
Positive control:
No positive control was used in this study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Details not reported.

DETAILED CLINICAL OBSERVATIONS: Details not reported.

BODY WEIGHT: Body weights were recorded at dosing and just before necropsy.

FOOD CONSUMPTION: Details not reported.

FOOD EFFICIENCY: Details not reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Details not reported.

OPHTHALMOSCOPIC EXAMINATION: Details not reported.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of study only
- Anaesthetic used for blood collection: Yes (Nembutal 100 mg/kg i.p.)
- Animals fasted: Not specified
- How many animals: 5 animals/dose
- Parameters examined.: Hematology analysis included measurement of the following parameters: erythrocyte, platelet and leukocyte counts, hematocrit, hemoglobin, mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of study only.
- Animals fasted: Not specified
- How many animals: 5 animals/dose
- Parameters examined: Serum chemistry analysis included measurement of the following parameters: albumin, total protein, urea nitrogen, creatinine, glucose, cholesterol, triglyceride, total bile acids, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, and 5’-nucleotidase

URINALYSIS: Details not reported.

NEUROBEHAVIOURAL EXAMINATION: Details not reported.

IMMUNOLOGY: Details not reported.

OTHER:
Forestomach and liver-cell proliferation were assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
At necropsy tissues were examined for gross lesions.

HISTOPATHOLOGY: Yes.
Sections of stomach, liver and kidney were fixed in 10 % neutral buffered formalin before H&E staining.
The remaining forestomach, glandular stomach and liver were then snap frozen on dry ice or in liquid nitrogen and stored until DNA isolation.
Other examinations:
No details reported.
Statistics:
Two approaches were used to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Body-weight data, which have approximately normal distributions, were analyzed using the parametric multiple comparison procedures of Williams and Dunnett. Hematology, clinical chemistry, cell proliferation, and cumulative percentage body-weight–change data, which typically have skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley and Dunn. Jonckheere’s test was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-response (Dunnett’s or
Dunn’s test). Trend-sensitive tests were used when Jonckheere’s test was significant at p < .01.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 rat (Control group) was killed on the first day of the study due to a dosing error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was observed in rats at 100 mg/kg bw/day (highest dose tested) for 4 weeks.
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
No details reported.
Food efficiency:
not specified
Description (incidence and severity):
No details reported.
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
No details reported.
Ophthalmological findings:
not examined
Description (incidence and severity):
No details reported.
Haematological findings:
no effects observed
Description (incidence and severity):
No effect on haematological parameters was reported.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effect on clinical chemistry was reported.
Urinalysis findings:
not examined
Description (incidence and severity):
No details reported
Behaviour (functional findings):
not examined
Description (incidence and severity):
No details reported.
Immunological findings:
not examined
Description (incidence and severity):
No details reported.
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
No details reported.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Lesions were observed only in the stomachand increased both in severity and incidence with dose.
Oedema in the stomach was observed in 2/5 rats at 30 mg/kg bw/day (mid -dose) and in most animals at 100 mg/kg bw/day
Neuropathological findings:
not examined
Description (incidence and severity):
No details reported.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic lesions were confined to the forestomach.

Exposure to 10 mg/kg for 4 weeks resulted in minimal, multifocal epithelial hyperplasia. Following exposure to 30 mg/kg, there was mild-to-moderate, multifocal-to-diffuse epithelial hyperplasia. Exposure to 100 mg/kg resulted in either moderateto- severe diffuse epithelial hyperplasia in 4 rats or severe diffuse hyperplasia (epithelization) with dysplasia in 1 rat.Damage to the gastric limiting ridge in exposed rats was not different from damage to the forestomach in these animals.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No details reported.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Positive trends in forestomach cell proliferation were observed. Increased were significant at 30 or 100 mg/kg bw/day (p<0.05) or 100 mg/kg bw/day (p<0.01).
Cell proliferation was not significantly increased in the liver at any dose level.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other:

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
other: forestomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
The only observed effects throughout the study were adverse lesions to the forestomach, thought to be due, primarily, to an irritation mode of action, with damage followed by regeneration/increased proliferation of stomach cells. Adverse changes to the forestomach are not considered to be applicable to humans, and sot therefore the NOAEL is determined to be 300 mg/kg bw/day (the highest dose tested), in the absence of systemic toxicity.
Executive summary:

The test substance was administed to F344 rats (5 males/dose), 5 days per week for 4 weeks. The only observed effects throughout the study were adverse lesions to the forestomach.The authors reported that this observation provides evidence that the test substance acts primarily through an irritation mode of action, with damage followed by regeneration/increased proliferation of stomach cells. Adverse changes to the forestomach are not considered to be applicable to humans, and so therefore the NOAEL is determined to be 300 mg/kg bw/day (the highest dose tested; approximately 214 mg/kg bw/day when adjusted for the 5 day/week dosing regimen), based on the absence of systemic toxicity.

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