Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 April 2019 - 24 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Source:
Envigo RMS B.V., Inc
Postbus 6174
5960 AD Horst / The Netherlands


Age (beginning of treatment): 15-16 weeks


Identification:
The animals were distributed into the test groups at random. If possible, all animals belonging to the same experimental group were kept in one cage. The animals were individually marked by indelible ink markings on the tail. A colour-coded card was prepared for each project, giving details of the test type, project number, treatment start, dose level, sex and number of animals.


Acclimatization:
At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


Housing:
During the exposure period: single
During the acclimation phase and after the exposure period: groups of up to three rats (of the same sex and dose group)


Cage Type: Makrolon Type IV, with wire mesh top


Bedding: granulated soft wood bedding


Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum


Water: tap water, ad libitum


temperature: 22 +/- 2°C


relative humidity: approx. 45-65 (with the aim of 50 – 60%)


artificial light: 6.00 a.m. - 6.00 p.m.


Environmental enrichment: provided throughout the study period (e.g., wooden chew blocks, fun tunnels or suitable nesting material)

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Preparation of Animals
- The day before dose application, the fur was removed from the dorsal region and both flanks of each animal using veterinary clippers. Care was taken to avoid abrading the skin.
- Prior to the start of the test the animals were weighed and the individual dose to be administered was adjusted to the animal’s body weight.

Study Substance
Batch: 20181120
Purity: 100%
Physical state, appearance: clear liquid
Expiration Date: 20 November 2021
Storage: Room Temp

Study Substance Preparation
- The study substance was used undiluted, as supplied, using a variable dose volume to achieve the required dosage. The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level.
-Study substance formulations were freshly prepared on the day of dosing, issued at room temperature and administered as soon as possible (within 4 hours of preparation).


Dose Administration
The study substance was applied evenly to an area of clipped skin equivalent to approximately 10% of the total body surface area. The site of application was covered with a gauze dressing backed with semi-occlusive surgical tape.

Exposure Period
-The exposure period was twenty-four hours, then the dressings were carefully removed. Residual study substance was removed by a cotton wool tissue soaked in water to remove any residual test item.
-After removal of the dressings and subsequently for 14 days, the test sites were examined for evidence of primary irritation and scored.
Duration of exposure:
24 hours
Doses:
2000 mg/kg
No. of animals per sex per dose:
n=3 females at 2000 mg/kg
Control animals:
not required
Details on study design:
Dose administration was once dermal (topical).

Based on available information on the toxicity of the study substance, 2000 mg/kg was chosen as the initial starting dose, since a severe toxicity which may necessitate humane euthanasia was not expected at this dose level.

In a pilot experiment, one single animal was treated at 2000 mg/kg. In the absence of mortality or toxicity at a dose level of 2000 mg/kg, 2 additional animals were treated at this dose (n=3 total).

After removal of the dressings and subsequently for 14 days, the test sites were examined for evidence of primary irritation and scored based on 1) Erythem and Eschar Formation and 2) Edema Formation. Any other skin reactions, if present were also recorded.

Clinical observations and inspections for morbidity / mortality were performed five times within the first six hours after application (i.e., 30 minutes and 1 hour, 2 hours, 4 hours and 5 hours after dosing), thereafter once daily for 14 days.

All animals were observed for 14 days after dosing.

The nature and severity, where appropriate, of the clinical signs and the time were recorded at each observation for all individual animals. If applicable, the time of death/humane kill was recorded as precisely as possible. Observations included changes in the skin and fur, eyes and mucous membranes, and respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behavior pattern. Particular attention was directed to the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

Body weights were recorded on Day 0 (prior to dosing), Day 7, and 14, or (if applicable) at death (unscheduled).

At the end of the study the animals were killed by CO2 asphyxiation.

A gross necropsy was performed on all animals that died or were humanely killed during the study (if applicable) and at the end of the in-life part. This consisted of an external examination and opening of the abdominal and thoracic cavities. Any macroscopic abnormalities were recorded. Routinely no organs or tissues were retained.
Statistics:
Evaluation of data includes the identification of the number of animals that died during the study (or that were killed for humane reasons), and determination of the nature, severity, onset and duration of the toxic effects. If possible, the signs of toxic effects of evident toxicity are described. Evident toxicity refers to the toxic effects of sufficient severity that administration of the next higher dose level could result in development of severe signs of toxicity and probable mortality. Effects on body weights and abnormalities noted at necropsy were identified.

Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.

Results and discussion

Preliminary study:
In a pilot experiment, one single animal was treated at 2000 mg/kg. In the absence of mortality or toxicity at a dose level of 2000 mg/kg, 2 additional animals were treated at this dose (n=3 total).
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No deaths observed
Mortality:
None
Clinical signs:
There were no clinical signs of reaction to treatment throughout the study.
Body weight:
The animals showed expected gains or only a negligible loss/stagnation in body weight over the observation period.
Gross pathology:
Erythema (score 1 to 2), very minor eschar formation, and scaly skin was observed, as well as small scratches.
Other findings:
During necropsy, the content of the stomach of one animal had an acidic smell. No abnormalities were noted for the other animals.

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
The acute median lethal oral dose (LD50) to rats of the study substance was demonstrated to be greater than 2000 mg/kg body weight.

The study substance is included in Category 5/Unclassified, according to the Globally Harmonised System (GHS).
Executive summary:

The study was performed to assess the acute dermal toxicity of the study substance to the rat.

Methods

Initially, one female animal was given a single, 24 hour, semi‑occluded dermal application of the undiluted study substance to intact skin at a dose level of 2000 mg/kg body weight. Based on the results of the initial test, further two female animals were similarly treated. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Results

Mortality: There were no deaths.

Clinical Observations: There were no signs of systemic toxicity.

Dermal Irritation: Erythema (score 1 to 2) very minor eschar formation, and scaly skin was observed, as well as small scratches.

Body Weight: All animals showed no relevant loss or even gain in body weight.

Necropsy: The content of the stomach of one animal had an acidic smell. No abnormalities were noted for the other animals.

Conclusion

The acute dermal median lethal dose (LD50) to rats of the study substance was found to be greater than 2000 mg/kg body weight.

The study substance is included in Category 5/Unclassified according to the Globally Harmonised System (GHS).