Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 January 2016 and 10 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 420 using a fixed dose method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

EC Number:

943-623-1

Molecular formula:

C13 H24 O

IUPAC Name:

Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

Test material form:

other: Liquid

Details on test material:

Identification: IFF 215 (Floriane)
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Justification
Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.

Animal Welfare
The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo - Shardlow policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the study may be reviewed, as part of the Envigo - Shardlow Ethical Review Process.
The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.

Animal Information
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the mean body weight at the start of treatment.

Animal Care and Husbandry
The animals were housed in groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

Test Item Preparation and Analysis
For the purpose of the 2000 mg/kg dose level the test item was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level. For the purpose of the 300 mg/kg dose level the test item was freshly prepared, as required, as a solution in arachis oil BP. Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Study Design
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.
A single animal was treated as follows:
Dose level: 300 mg/kg
Concentration: 30 mg/mL
Dose volume: 10 mL/kg
Number of rats: 1 female

In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated as follows:
Dose level: 2000 mg/kg
CSpecific gravity: 0.922
Dose volume: 2.17 mL/kg
Number of rats: 1 female

In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
Dose level: 2000 mg/kg
CSpecific gravity: 0.922
Dose volume: 2.17 mL/kg
Number of rats: 4 females

A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.
All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.

Doses:

300 and 2000 mg/kg

No. of animals per sex per dose:

A single animal was treated at 300 mg/kg.
In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated at 2000 mg/kg.
In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of four animals were treated at 2000 mg/kg.
A total of five animals were therefore treated at a dose level of 2000 mg/kg.

Control animals:

no

Details on study design:

Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily.
Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: There was no mortality during the study.

Mortality:

Dose Level - 300 mg/kg
There was no mortality.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
There were no deaths.

Clinical signs:

Dose Level - 300 mg/kg
No signs of systemic toxicity were noted during the observation period.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
No signs of systemic toxicity were noted in the initial treated animal during the observation period.
Signs of systemic toxicity noted in the additional treated animals were hunched posture, ataxia, pilo erection, tiptoe gait, increased salivation, noisy respiration, sneezing and red/brown staining around the eyes. These four animals appeared normal 2 days after dosing.

Body weight:

Dose Level - 300 mg/kg
The animal showed expected gains in body weight over the observation period.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
Animals showed expected gains in body weight over the observation period, except for one animal which showed expected gain in body weight during the first week but no gain in body weight during the second week.

Gross pathology:

Dose Level - 300 mg/kg
No abnormalities were noted at necropsy.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
No abnormalities were noted at necropsy.

Any other information on results incl. tables

Individual Clinical Observations and Mortality Data -300mg/kg

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
300	1-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

Individual Body Weights and Body Weight Changes -300mg/kg

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight Gain (g) During Week
		0	7	14	1	2
300	1-0 Female	179	191	193	12	2

Individual Necropsy Findings -300 mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
300	1-0 Female	Killed Day 14	No abnormalities detected

Individual Clinical Observations and Mortality Data -2000mg/kg

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	2-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-0 Female	0	0	H	HA	HAPWtSe	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-1 Female	0	0	H	HAWt	HAWt	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-2 Female	0	0	H	HAWt	HWt	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-3 Female	0	RnS	HSRnRs	HARnRs	HRs	0	0	0	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

H = Hunched posture,

A =  Ataxia

P =   Pilo‑erection

Wt =Tiptoe gait

S =   Increased salivation

Rn =Noisy respiration,

Rs =Sneezing

Se = red/brown staining around the eyes

Individual Body Weights and Body Weight Changes -2000mg/kg

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight Gain (g) During Week
		0	7	14	1	2
2000	2-0 Female	155	166	179	11	13
	3-0 Female	173	186	201	13	15
	3-1 Female	178	190	202	12	12
	3-2 Female	163	171	186	8	15
	3-3 Female	176	187	187	11	0

Individual Necropsy Findings -2000mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	2-0 Female	Killed Day 14	No abnormalities detected
	3-0 Female	Killed Day 14	No abnormalities detected
	3-1 Female	Killed Day 14	No abnormalities detected
	3-2 Female	Killed Day 14	No abnormalities detected
	3-3 Female	Killed Day 14	No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System - Category 5).

Executive summary:

The acute oral toxicity of the test substance, IFF 215 (Floriane), was assessed according to OECD Test Guideline 420 using a fixed dose method. The LD50 was estimated to be greater than 2000 mg/kg body weight.