3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 15 March 2016 - Experimental completion date 05 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

1

Reference substance name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

EC Number:

814-345-7

Cas Number:

2003244-43-5

Molecular formula:

C14H21N

IUPAC Name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Details on test material:

- Name of test material (as cited in study report): ES421 Pinyl Nitrile- Molecular weight (if other than submission substance): 203 g/mol- Physical state: Crystallized white powder- Analytical purity: 98.08% (per Certificate of Analysis)- Composition of test material, percentage of components: 2-Norpinene-2-propionitrile, alpha, alpha,6,6-tetramethyl- Purity test date: 08 February 2016- Lot/batch No.: SM15077102- Storage condition of test material: 2-8°C, protected from light

Specific details on test material used for the study:

Physical state/Appearance: white solid
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:

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.
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 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

Remarks:

Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.

Details on oral exposure:

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 group to confirm the survival of the previously dosed animals.

Doses:

300 mg/kg was chosen as the starting dose.
2000 mg/kg.

No. of animals per sex per dose:

3 animals for 300 mg/kg.
6 animals for 2000 mg/kg.

Control animals:

no

Details on study design:

Groups of treated animals were treated as shown on Table A.
The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for up to 14 days.
Individual body weights were recorded prior to dosing and 7 and 14 days after treatment or at death.
At the end of the observation period the surviving animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. 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.

Mortality:

Two animals treated at a dose level of 2000 mg/kg were killed for humane reasons, 1 or 2 days after dosing, due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence. See Table 1.

Clinical signs:

Signs of systemic toxicity noted in four animals treated at a dose level of 2000 mg/kg included hunched posture, pilo-erection, ptosis, increased salivation, labored respiration, decreased respiratory rate, diarrhea, tiptoe gait and lethargy.
There were no signs of systemic toxicity noted in two animals treated at a dose level of 2000 mg/kg and all animals treated at a dose level of 300 mg/kg. See Tables 2 and 3.

Body weight:

Surviving animals showed expected gains in body weight over the observation period. See Tables 4 and 5.

Gross pathology:

Abnormalities noted at necropsy of animals that were humanely killed were pale liver, or patchy pallor of the liver, hemorrhagic gastric mucosa and blood filled bladder. No abnormalities were noted at necropsy of animals that were killed at the end of the study. See Tables 6 and 7.

Any other information on results incl. tables

Table 1: Mortality Data

Dose Level mg/kg	Sex	Number of Animals Treated	Deaths During Day of Dosing (Hours)				Deaths During Period After Dosing (Days)								Deaths
			½	1	2	4	1	2	3	4	5	6	7	8-14
300	Female	3	0	0	0	0	0	0	0	0	0	0	0	0	0/3
2000	Female	3	0	0	0	0	1*	0	0	0	0	0	0	0	1/3
	Female	3	0	0	0	0	0	1*	0	0	0	0	0	0	1/3

*=   Animal killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence

Table 2: Individual Clinical Observations - 300 mg/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
	1-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2 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

Table 3: Individual Clinical Observations - 2000 mg/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	H	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	PtSPHD RlRdX*
	2-2 Female	0	0	0	0	HPWt	H	0	0	0	0	0	0	0	0	0	0	0	0
	3-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-2 Female	0	0	0	0	0	PtSPHD RlRdLX*

0= No signs of systemic toxicity                 D = Diarrhea                     H = Hunched posture                    L = Lethargy                                   P = Pilo‑erection

Pt = Ptosis           Rd = Decreased respiratory rate                                 Rl = Labored respiration                S = Increased salivation               Wt = Tiptoe gait

X* = Killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence

Table 4: Individual Body Weights and Body Weight Changes - 300 mg/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	171	182	188	11	6
	1-1 Female	192	217	226	25	9
	1-2 Female	178	191	201	13	10

Table 5: Individual Body Weights and Body Weight Changes - 2000 mg/kg

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight (g) at Death	Body Weight Gain (g) During Week
		0	7	14		1	2
2000	2-0 Female	180	197	214		17	17
	2-1 Female	167	-	-	154	-	-
	2-2 Female	183	196	204		13	8
	3-0 Female	178	188	208		10	20
	3-1 Female	159	168	174		9	6
	3-2 Female	174	-	-	150	-	-

 

Table 6: 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
	1-1 Female	Killed Day 14	No abnormalities detected
	1-2 Female	Killed Day 14	No abnormalities detected

 

Table 7: Individual Necropsy Findings - 2000 mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	2-0 Female	Killed Day 14	No abnormalities detected
	2-1 Female	Humanely killed Day 1	Liver: pale Gastric mucosa: hemorrhage Bladder: blood filled
	2-2 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	Humanely killed Day 2	Liver: patchy pallor Gastric mucosa: hemorrhage Bladder: blood filled

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

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, >2000 - 5000 mg/kg body weight).

Executive summary:

The acute oral median lethal dose (LD₅₀) of ES421 Pinyl Nitrile in the Wistar strain rat was found to be greater than 2000 mg/kg body weight according to OECD Test Guideline 423 using the acute oral toxicity method.