5H-Cyclopenta[h]quinazoline, 6,6a,7,8,9,9a-hexahydro-7,7,8,9,9-pentamethyl-

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 23 April 2014 and 28 May 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

EC Number:

801-694-5

Cas Number:

1392325-86-8

Molecular formula:

C16H24N2

IUPAC Name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

Test material form:

other: Solid

Details on test material:

Identification: IFF TM 12-206 (FRET 10-0199)
Description: White solid block
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:

Female Wistar (RccHan:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., 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 five 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 eight to twelve weeks of age. The body weight variation did not exceed ±20% of the body weight of the initially dosed animal.

The animals were housed in groups of up to five in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., 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 twelve hours continuous light (06:00 to 18:00) and twelve 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:

DMSO

Details on oral exposure:

For the purpose of the study the test item was freshly prepared, as required, as a solution in dimethyl sulphoxide. Dimethyl sulphoxide was used because the test item did not dissolve/suspend in distilled water or arachis oil BP.

The test item was formulated within two 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.

Doses:

Following a sighting test at dose levels of 2000 mg/kg and 300 mg/kg, a further group of four fasted females was given a single oral dose of test item, as a solution in dimethyl sulphoxide, at a dose level of 2000 mg/kg body weight. Due to mortalities and signs of systemic toxicity at a dose level of 2000 mg/kg, an additional group of five animals was similarly treated at a dose level of 300 mg/kg body weight.

No. of animals per sex per dose:

Sighting test at dose levels of 2000 mg/kg and 300 mg/kg: One per dose
Main test at dose levels of 2000 mg/kg: Four per dose
Main test at dose levels of 300 mg/kg: Five per dose

Control animals:

no

Details on study design:

Using available information on the toxicity of the test item, 300 mg/kg was chosen as the starting dose.

A single animal was treated as follows:
300 mg/kg (at a concentration of 30 mg/mL) at a dose volume of 10 mL/kg administered to 1 female rat.

In the absence of mortality at a dose level of 300 mg/kg, an additional animal was treated as follows:
2000 mg/kg (at a concentration of 200 mg/mL) at a dose volume of 10 mL/kg administered to 1 female rat.

In the absence of mortality at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
2000 mg/kg (at a concentration of 200 mg/mL) at a dose volume of 10 mL/kg administered to 4 female rats.

A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

Due to mortality and signs of systemic toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
300 mg/kg (at a concentration of 30 mg/mL) at a dose volume of 10 mL/kg administered to 5 female rats.

A total of six animals were therefore treated at a dose level of 300 mg/kg in the study.

Due to a technician error and Study Director oversight, the final group of animals, treated at a dose level of 300 mg/kg body weight, comprised of five animals instead of the required four animals meaning that a total of six animals were treated at this dose level instead of the required five. This deviation was considered not to affect the purpose or integrity of 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 level to confirm the survival of the previously dosed animals.

Clinical observations were made 0.5, 1, 2, and 4 hours after dosing and then daily for up to fourteen 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 or at death.

At the end of the observation period the surviving 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:

300 - 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Dose Level - 2000 mg/kg
Two animals were found dead 1 or 4 days after dosing. One animal was killed for humane reasons, 6 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.

Dose Level - 300 mg/kg
One animal was killed for humane reasons, during the day of dosing, due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence.

Clinical signs:

other: Dose Level - 2000 mg/kg Signs of systemic toxicity noted were hunched posture, lethargy, pilo erection, decreased respiratory rate, labored respiration, splayed or tiptoe gait, occasional body tremors, ataxia, dehydration, diarrhea and emaciation. Dose

Gross pathology:

Dose Level - 2000 mg/kg
Abnormalities noted at necropsy of animals that died or were humanely killed during the study were dark liver or patchy pallor of the liver, dark kidneys, solid substance present in the stomach, epithelial sloughing of the gastric mucosa and hemorrhagic non glandular epithelium of the stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Dose Level - 300 mg/kg
Clear liquid or solid substance present in the stomach and thickened non glandular epithelium were noted at necropsy of the animal that was humanely killed during the study. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Any other information on results incl. tables

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	H	HLRd	HPL	HLP	HPWt	H	H	H	0	0	0	0	0	0	0	0	0	0
	3-0 Female	HWs	WsToL	WsL	WsL	HLA RdDh	HRl	HRl	H	H	HRdEm DDhX*
	3-1 Female	Ws	WsToL	WsL	WsL	HLA RdDh	HRl	HRl	H	H	0	0	0	0	0	0	0	0	0
	3-2 Female	Ws	HLA	HLA	WsL	HLA RdDh	HRlRd	HRlRd	X
	3-3 Female	WsH	HLA	HLA	HLA	HLA RdDhX

0= No signs of systemic toxicity                      

H = Hunched posture                        

L = Lethargy                        

P = Pilo‑erection                 

A = Ataxia

Rd = Decreased respiratory rate                      

Rl = Labored respiration                  

Ws = Splayed gait              

Wt = Tiptoe gait                  

To = Occasional body tremors

Dh = Dehydration                                             

D = Diarrhea                                       

Em = Emaciation                               

X = Animal dead

X* = 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

Individual Body Weights and Body Weight Changes -2000mg/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	171	158	169		-13	11
	3-0 Female	158	-	-	120	-	-
	3-1 Female	171	164	192		-7	28
	3-2 Female	163	-	-	134	-	-
	3-3 Female	155	-	-	135	-	-

-=       Animal dead

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
	3-0 Female	Humanely killed Day 6	Liver: dark Kidneys: dark Gastric mucosa: epithelial sloughing
	3-1 Female	Killed Day 14	No abnormalities detected
	3-2 Female	Found dead Day 4	Liver: dark Kidneys: dark
	3-3 Female	Found dead Day 1	Liver: patchy pallor Stomach: solid substance present Non-glandular epithelium of the stomach: hemorrhage

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	HL	HLP	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4-0 Female	0	H	H	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4-1 Female	HWt	HWt	HWt	H	H	H	0	0	0	0	0	0	0	0	0	0	0	0
	4-2 Female	H	HWt	HWsL	PrRdRl HoX*
	4-3 Female	0	H	H	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	4-4 Female	H	H	H	H	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0= No signs of systemic toxicity                     

H = Hunched posture                        

L = Lethargy                                        

P = Pilo‑erection        

Pr = Prostration

Ho = Hypothermia                                             

Rd = Decreased respiratory rate      

Rl = Labored respiration                  

Ws = Splayed gait      

Wt = Tiptoe gait

X* = 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

Individual Body Weights and Body Weight Changes -300mg/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
300	1-0 Female	167	183	196		16	13
	4-0 Female	157	171	183		14	12
	4-1 Female	178	200	218		22	18
	4-2 Female	178	-	-	164	-	-
	4-3 Female	167	190	199		23	9
	4-4 Female	175	195	211		20	16

-=       Animal dead

Individual Necropsy Findings -300mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
300	1-0 Female	Killed Day 14	No abnormalities detected
	4-0 Female	Killed Day 14	No abnormalities detected
	4-1 Female	Killed Day 14	No abnormalities detected
	4-2 Female	Humanely killed Day 0	Stomach: clear liquid present Non-glandular epithelium of the stomach: thickened
	4-3 Female	Killed Day 14	No abnormalities detected
	4-4 Female	Killed Day 14	No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:

other: Harmful

Remarks:

in accordance with EU CLP (EC no. 1272/2008 and its amendments)

Conclusions:

The acute oral LD50 for the substance in male and female rats was determined to be between 300 and 2000 mg/kg bw (and roughly estimated to be 1850).

Executive summary:

The substance was tested in an acute oral toxicity test, using the fixed dose method (OECD TG 420) at doses of 2000 and 300 mg/kg bw. At 2000 mg/kg bw, mortality occurred in two out of five animals, while a third animal was killed for humane reasons due to severe clinical signs of toxicity. At 300 mg/kg bw, one animal was killed out of humane reasons due to severe clinical signs of toxicity. Clinical signs at 2000 mg/kg bw included hunched posture, lethargy, pilo-erection, decreased respiratory rate, labored respiration, splayed or tiptoe gait, occasional body tremors, ataxia, dehydration, diarrhea and emaciation. Macroscopy in animals that either died or were humanely killed during the study at 2000 mg/kg bw showed a dark liver or patchy pallor of the liver, dark kidneys, solid substance present in the stomach, epithelial sloughing of the gastric mucosa and hemorrhagic non-glandular epithelium of the stomach. At 300 mg/kg bw, clinical signs included hunched posture, lethargy, pilo-erection. For the animal that was humanely killed during the study, clinical signs included a decreased respiratory rate, labored respiration, splayed gait, prostration and hypothermia. Macroscopy in the animal that was humanely killed during the study at 300 mg/kg bw showed presence of a clear liquid or solid substance in the stomach and a thickened non-glandular epithelium. All surviving animals appeared normal at the end of the study and no abnormalities were noted for these animals during macroscopy. The acute oral LD50 resulted in 300 -2000 mg/kg bw and is roughly estimated to be 1850 mg/kg bw.