((4S,4aR,7S,7aR)-4,7-dimethylhexahydrocyclopenta[c]pyran-1(3H)-one

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 20 - July 7, 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 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

up-and-down procedure

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

N/A

Molecular formula:

C10H16O2

Test material form:

liquid

Remarks:

Colorless

Details on test material:

Test substance was expected to be stable for the duration of testing.

Specific details on test material used for the study:

- lot number of test material:
CH-DA-01-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Stored refrigerated

- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
Test substance was expected to be stable for the duration of testing.

- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
The test substance was applied as received

- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
The test substance was applied as received

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing (e.g. warming, grinding):
Mixed well prior to use

- Preliminary purification step (if any):
None

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 9 - 12 weeks
- Weight at study initiation: 206 - 264 gram females
- Housing:
The animals were housed in caging which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Animals were group housed, except on the day of application, at which time they were singly housed until the animals were deemed acceptable, based on observations, to return to group housing. Enrichment (e.g., toy) was placed in each cage and litter was changed at least once per week.

- Diet (e.g. ad libitum):
Envigo Teklad Global 16% Protein Rodent Diet® #20 16. The diet was available ad libitum.

- Water: Filtered tap water was supplied ad libitum

- Acclimation period: 12 - 34 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 53 - 68
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DOSAGE PREPARATION:
Individual doses were calculated based on the initial body weights, taking into account the density of the test substance

Doses:

175, 550, 1750, 5000 mg/kg

No. of animals per sex per dose:

8 females, divided as follows:
175 mg/kg: 1
550 mg/kg: 1
1750 mg/kg: 3
5000 mg/kg:3

Details on study design:

- Duration of observation period following administration:
14 days

- Frequency of observations and weighing:
mortality, signs of gross toxicity, and behavioral changes approximately 30 minutes post-dosing, during the first several hours post-dosing and at least once daily thereafter for 14 days after dosing or until death occurred. Individual body weights prior to test substance administration and on Days 7 and 14 following dosing or after death.

- Necropsy of survivors performed:
yes

- Clinical signs including body weight
gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma. Individual body weights of the animals were recorded.

- Other examinations performed:
Gross necropsies were performed on all decedents and euthanized animals. Tissues and organs of the thoracic and abdominal cavities were examined.

Statistics:

Statistical analysis was performed for the calculation of the mean density value for dosing. In
addition, the Acute Oral Toxicity (Guideline 425) Statistical Program (Westat 2001) was used for
all data analyses including: dose progression selections, stopping criteria determinations and/or
LD50 and confidence limit calculations.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

3 129 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Approximate 95% confidence interval of 1750 mg/kg (lower) to 5000 mg/kg (upper).

Mortality:

At the highest dose, all animals died within five hours of test substance administration. No deaths were observed at lower doses.

Clinical signs:

other: 175 mg/kg: irregular respiration and ataxia. 550 mg/kg: hypoactive and exhibited irregular respiration. 1750 mg/kg: one female was hypoactive and two females exhibited irregular respiration, ataxia, tremors, piloerection and/or reduced fecal volume. 5000

Gross pathology:

Gross necropsy of the decedents revealed distention of the stomach and/or a fluid-filled stomach.

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Under the conditions of this study, the acute oral LD50 of Dihydronepetalactone is estimated to be
3129 mg/kg of body weight (based on an assumed sigma of 0.5) in female rats with an
approximate 95% confidence interval of 1750 mg/kg (lower) to 5000 mg/kg (upper).

Executive summary:

An acute oral toxicity test (Up and Down Procedure) was conducted with rats to determine the potential for Dihydronepetalactone to produce toxicity from a single dose via the oral route.

Based on a limit dose of 5000 mg/kg, a Main Test was conducted using a default starting dose level of 175 mg/kg which was administered to one healthy female rat by oral gavage. Following the Up and Down procedure, seven additional animals were dosed at levels of 550, 1750 or 5000 mg/kg. Females were selected for the test because they are frequently more sensitive to the toxicity of test compounds than males. All animals were observed for mortality, signs of gross toxicity, and behavioral changes at least once daily for 14 days after dosing or until death occurred. Body weights were recorded prior to administration (initial) and again on Days 7 and 14 (terminal) following dosing or after death. Necropsies were performed on all animals.

Under the conditions of this study, the acute oral LD50 of the test substance is estimated to be 3129 mg/kg of body weight (based on an assumed sigma of 0.5) in female rats with an approximate 95% confidence interval of 1750 mg/kg (lower) to 5000 mg/kg (upper).