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Description of key information

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.


An acute dermal toxicity test was conducted with rats to determine the potential for Dihydronepetalactone to produce toxicity from a single topical application. Under the conditions of this study, the single dose acute dermal LD50 of the test substance is greater than 5000 mg/kg of body weight in male and female rats.


Five thousand milligrams of the test substance per kilogram of body weight was applied to the skin of ten healthy rats for 24 hours. The animals were observed for mortality, signs of gross toxicity, and behavioral changes at least once daily for 14 days. Body weights were recorded prior to application (initial) and again on Days 7 and 14 (terminal). Necropsies were performed on all animals at terminal sacrifice.


All animals survived test substance administration, gained body weight, and appeared active and healthy during the study. There were no signs of gross toxicity, dermal irritation, adverse clinical effects, or abnormal behavior. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.


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).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
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
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
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
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
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.
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:
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 mg/kg: Prior to death, two animals exhibited irregular respiration and prone posture.
Body weight:
175 mg/kg: weight gain;
550 mg/kg: weight gain;
1750 mg/kg: weight gain;
5000 mg/kg: death within 5 hours of substance administration
Gross pathology:
Gross necropsy of the decedents revealed distention of the stomach and/or a fluid-filled stomach.
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).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
3 129 mg/kg bw

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 19-June 2, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes
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
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10 weeks
- Weight at study initiation: 330-3 59 grams and females 222-245 grams
- 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: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 34 - 68
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: 2 inches x 3 inches
- % coverage: approximately 10% of the body surface
- Type of wrap if used: 3-inch Durapore tape

REMOVAL OF TEST SUBSTANCE
- Washing: 3% soap solution followed by tap water and a clean paper towel
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied: 5000 mg/kg bw
- Concentration (if solution): applied as received

VEHICLE
No vehicle. Substance applied as received.
Duration of exposure:
24 hours
Doses:
- other: 5000 mg/kg bw
No. of animals per sex per dose:
5
Details on study design:
- Duration of observation period following administration:
14 days post exposure

- Frequency of observations and weighing:
observed for mortality, signs of gross toxicity, and behavioral changes during the first several hours after application, after patch removal, and then at least once daily thereafter for 14 days. Individual body weights of the animals were recorded prior to test substance application (initial) and again on Days 7 and 14 (terminal).

- 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.

- Other examinations performed:
Gross necropsies were performed on all animals. Tissues and organs of the thoracic and abdominal cavities were examined.
Statistics:
Limited to the calculation of the mean density value for dosing.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No mortality up to the highest dose tested.
Mortality:
No mortalities
Clinical signs:
No clinical signs
Body weight:
All animal gained body weight
Gross pathology:
No signs of gross toxicity
Other findings:
No signs of dermal irritation, adverse clinical effects, or abnormal behavior.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the single dose acute dermal LD50 of Dihydronepetalactone is
greater than 5000 mg/kg of body weight in male and female rats.
Executive summary:

An acute dermal toxicity test was conducted with rats to determine the potential for Dihydronepetalactone to produce toxicity from a single topical application. Under the conditions of this study, the single dose acute dermal LD50 of the test substance is greater than 5000 mg/kg of body weight in male and female rats.



Five thousand milligrams of the test substance per kilogram of body weight was applied to the skin of ten healthy rats for 24 hours. The animals were observed for mortality, signs of gross toxicity, and behavioral changes at least once daily for 14 days. Body weights were recorded prior to application (initial) and again on Days 7 and 14 (terminal). Necropsies were performed on all animals at terminal sacrifice.


 


All animals survived test substance administration, gained body weight, and appeared active and healthy during the study. There were no signs of gross toxicity, dermal irritation, adverse clinical effects, or abnormal behavior. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
> 5 000 mg/kg bw

Additional information

Justification for classification or non-classification

Adverse effects are observed for the substance Dihydronepetalactone in both the Acute toxicity oral and dermal studies.  However, the effects were classified as Category 5 under GHS and it does not meet the criteria for classification according to Regulation (EC) No 1272/2008

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