[No public or meaningful name is available]

Endpoint summary

Description of key information

The oral LD50 value of Red HF2 in Wistar Han rats was established to exceed 2000 mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed 5000 mg/kg body weight.
Based on these results, Red HF2 does not have to be classified and has no obligatory labelling requirement for acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2021) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

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:

04 October - 01 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study conducted to current guideline

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines (2000), including the most recent revisions.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

No further details in the study report

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Animals: 6 Females (nulliparous and non-pregnant). Each dose group consisted of 3 animals.
Age at the Initiation of Dosing: Young adult animals (approximately 9 weeks old) were selected.
Weight at the Initiation of Dosing: 164 to 187 g.

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as recognized by international guidelines as a recommended test system. The test method and number of animals were based on the test guidelines.

The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using a subcutaneously implanted electronic identification chip.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

Husbandry
Housing
On arrival and following assignment to the study, animals were group housed (up to 3 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized wooden fibers as bedding material equipped with water bottles. The room in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 20 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21 to 22°C with an actual daily mean relative humidity of 50 to 68%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Vehicle
Identification: Corn oil
Supplier: Sigma-Aldrich, Steinheim, Germany
Specific gravity: 0.92

Rationale for Vehicle
Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw Data of these trials will be retained by the Test Facility

Justification of Route and Dose Levels
The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test material.
The dose levels were based on the OECD test guidelines and were selected from the series 5 (lowest dose level), 50, 300 and 2000 (highest dose level) mg/kg body weight. The starting dose level should be the one that is likely to produce mortality in at least some of the animals and was selected based on available toxicity data of the test material.

Administration of Test material
A single dose of test material was administered to the appropriate animals by oral gavage on Day 1, using a syringe with a plastic gavage cannula attached.
The dose volume for each animal was based on the body weight measurement prior to dosing.
A dose volume of 10 mL/kg body weight was used for each dose.
The dosing formulations were stirred continuously during dose administration.
Animals were deprived of food overnight (for a maximum of 20 hours) prior to dosing and until 3-4 hours after administration of the test material. Water was available.

Doses:

2000mg/kg bw

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

Preparation of Test Material
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. To liquify the test material, it was heated for approximately 90 minutes at 64°C prior to the preparation of the formulations.
The formulations were heated to a maximum of 64°C for maximally 75 minutes. The formulations were released for dosing when they obtained a temperature of 40°C or lower.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
Any residual volumes were discarded.

Sample Collection and Analysis
Analysis of test material in vehicle for concentration, stability, homogeneity was not performed.

In - Life Procedures, Observations, and Measurements
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Post-dose Observations
Post-dose observations for clinical signs were performed at periodic intervals on the day of dosing (at least three times) and once daily thereafter. The observation period was 14 days. All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals for the first hour after dosing.

Body Weights
Animals were weighed individually on Day 1 (pre-dose), 8 and 15. A fasted weight was recorded on the day of dosing.

Terminal Procedures
All animals were sacrificed by oxygen/carbon dioxide procedure at the end of the observation period. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No premature mortality occurred.

Clinical signs:

other:

Body weight:

other body weight observations

Remarks:

The body weight gain shown by the animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain.

Gross pathology:

No observations found

Individual Clinical Observations

Group 1 Sex: Female 2000 mg/kg
	Observation Type: All Types	Day(s) Relative to Group Start Date
		1 0h	1 2h	1 4h	2	3	4	5	6	7	8	9	10	11	12	13	14	15
1	Hunched Posture	.	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Feces, Abnormal Color, Red, Group Housed	.	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
2	Hunched Posture	.	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Feces, Abnormal Color, Red, Group Housed	.	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
3	Hunched Posture	.	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Feces, Abnormal Color, Red, Group Housed	.	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.

 

 

Group 2 Sex: Female 2000 mg/kg
	Observation Type: All Types	Day(s) Relative to Group Start Date
		1 0h	1 2h	1 4h	2	3	4	5	6	7	8	9	10	11	12	13	14	15
4	Hunched Posture	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	.	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Eye Closed, Site Not Recorded, Partly	.	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Feces, Abnormal Color, Red, Group Housed	.	.	.	X	X	X	X	.	.	.	.	.	.	.	.	.	.
	Activity Decreased	.	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
5	Hunched Posture	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	.	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Feces, Abnormal Color, Red, Group Housed	.	.	.	X	X	X	X	.	.	.	.	.	.	.	.	.	.
6	Hunched Posture	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	.	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Feces, Abnormal Color, Red, Group Housed	.	.	.	X	X	X	X	.	.	.	.	.	.	.	.	.	.

X = Present

. = Not present

 

Individual Body Weights

Sex: Female Bodyweight (g)

 

 

2000 mg/kg Group 1	Day(s) Relative to Group Start Date
	1		8		15
1	187		201		227
2	165		186		195
3	174		186		207
Mean	175.3		191.0		209.7
SD	11.1		8.7		16.2
N		3		3		3

 

 

2000 mg/kg Group 2	Day(s) Relative to Group Start Date
	1		8		15
4	175		197		204
5	168		178		196
6	164		180		199
Mean	169.0		185.0		199.7
SD	5.6		10.4		4.0
N		3		3		3

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 value of Red HF2 in Wistar Han rats was established to exceed 2000 mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed 5000 mg/kg body weight.
Based on these results, Red HF2 does not have to be classified and has no obligatory labelling requirement for acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2021) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to assess the toxicity of the test material when administered in a single dose to female rats at one or more defined dosages. Furthermore, the results of the study allowed the test material to be ranked according to most classification systems, currently in use. This study should provide a rational basis for risk assessment in human.
The study was carried out in compliance with the guidelines described in:
● OECD No.423 (2001) "Acute Oral Toxicity, Acute Toxic Class Method".
● EC No 440/2008, part B: "Acute Oral Toxicity, Acute Toxic Class Method".
● EPA, OPPTS 870.1100 (2002), "Acute Oral Toxicity".
● JMAFF Guidelines (2000), including the most recent revisions.
Red HF2 was administered by oral gavage to two consecutive groups of three female Wistar Han rats at 2000 mg/kg body weight. Animals were subjected to daily observations for clinical signs, and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (Day 15).
Hunched posture and erected fur were observed in all animals on Day 1, but there was no premature mortality. Three out of six animals also showed hunched posture on Day 2 and one animal showed partly closed eyes and decreased activity on Day 1. Red feces were noted for three out of six animals on Day 1 and for the other three animals on Days 2-5.
The body weight gain shown by the animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain.
No abnormalities were found at macroscopic post mortem examination of the animals. The oral LD50 value of Red HF2 in Wistar Han rats was established to exceed 2000 mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed 5000 mg/kg body weight.
Based on these results, Red HF2 does not have to be classified and has no obligatory labelling requirement for acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2021) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

1