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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 September 2011 to 27 September 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The substance showed sensitisation reactivity without a concentration relation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline

EC Number:

801-093-8

Cas Number:

1315251-11-6

Molecular formula:

C16H22N2

IUPAC Name:

(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation 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 in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately 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.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50%, 25% or 10% w/w

No. of animals per dose:

5

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the substance, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the substance at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (days 1, 2, 3). The mouse was observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Local irritation was scored daily according to the scale included as Appendix 3. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on day 1 (prior to dosing) and on day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on day 1, post dose on day 3 and on day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods days 1 to 3 and days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Substance Administration
Groups of five mice were treated with the substance at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the substance would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the substance to the dorsal surface of each ear for three consecutive days (days 1, 2, 3). The substance formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner to act as a negative control.
The positive control group animals were similarly treated to the test animals, except that 25 µl of the positive control item, α-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration
Five days following the first topical application of the substance, vehicle or positive control item (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on days 1, 2 and 3 and on a daily basis on days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The body weight of each mouse was recorded on day 1 (prior to dosing) and day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 ml of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

16.1

Test group / Remarks:

10% test substance

Remarks on result:

other: 10%

Results 2

Key result

Parameter:

SI

Value:

16.88

Test group / Remarks:

25% test substance

Remarks on result:

other: 25%

Results 3

Key result

Parameter:

SI

Value:

9.15

Test group / Remarks:

50% test substance

Remarks on result:

other: 50%

Any other information on results incl. tables

Interpretation of Results

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of ³HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in ³HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in ³HTdR incorporation will be classified as a "non‑sensitiser".

Preliminary Screening Test

Clinical observations, body weight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

Light brown coloured residual test item on the ears was noted on Days 1 to 3. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in acetone/olive oil 4:1.

Main Test Estimation of the Proliferative Response of Lymph Node Cells The radioactive disintegrations per minute per animal and the stimulation index are given in Table 4.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	10% w/w in acetone/olive oil4:1	16.10	Positive
	25% w/w in acetone/olive oil4:1	16.88	Positive
	50% w/w in acetone/olive oil4:1	9.15	Positive
Positive Control Item	25% v/v in acetone/olive oil4:1	10.63	Positive

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

Light brown coloured residual test item on the ears was noted in animals treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1. 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

  

Bodyweights

Individual body weights and body weight changes for test and control animals are given in Table 6.

Body weight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
50	S-1	19	20	0	0Rt	0Rt	0Rt	0	0Rt	0	0	0

0=     No signs of systemic toxicity

Rt =     Light brown coloured residual test item on the ears

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration (% w/w) in acetone/olive oil 4:1

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

pre‑dose

post dose

post dose

left

right

left

right

left

right

left

right

left

right

left

right

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-1	0.230	0.230	0.275	0.270	0.245	0.235
overall mean (mm)		0.230		0.273		0.240
overall mean ear thickness change (%)		na		18.478		4.348

na=     Not applicable

Table 4              Individual Disintegrations per Minute and Stimulation Indices

Treatment Group	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle acetone/olive oil 4:1	1-1	1939.67	1356.89 (±386.52)	na	na
	1-2	1478.75
	1-3	915.04
	1-4	1307.22
	1-5	1143.79
Test Item 10% w/w in acetone/oliveoil 4:1	2-1	30263.05	21842.08*** (±9827.15)	16.10	Positive
	2-2	24595.09
	2-3	24112.69
	2-4	4817.71
	2-5	25421.84
Test Item 25% w/w in acetone/oliveoil 4:1	3-1	28659.34	22910.12*** (±3619.76)	16.88	Positive
	3-2	19227.22
	3-3	21852.20
	3-4	20962.72
	3-5	23849.12
Test Item 50% w/w in acetone/oliveoil 4:1	4-1	14643.56	12419.70* (±6409.89)	9.15	Positive
	4-2	16313.09
	4-3	17090.90
	4-4	1358.59
	4-5	12692.34
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	14532.58	14430.07* (±5754.04)	10.63	Positive
	5-2	13720.55
	5-3	8737.79
	5-4	23891.58
	5-5	11267.83

dpm=Disintegrations per minute

a=     Total number of lymph nodes per animal is 2

b=     Stimulation Index of 3.0 or greater indicates a positive result

na=     Not applicable

***=   Significantly different from control group p<0.001

*=      Significantly different from control group p<0.05

Table 5          Individual Clinical Observations and Mortality Data

Treatment Group	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle acetone/olive oil 4:1	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
	1-5	0	0	0	0	0	0	0	0	0
Test Item 10% w/w in acetone/oliveoil 4:1	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
	2-5	0	0	0	0	0	0	0	0	0
Test Item 25% w/w in acetone/oliveoil 4:1	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
	3-5	0	0	0	0	0	0	0	0	0
Test Item 50% w/w in acetone/oliveoil 4:1	4-1	0	0	0	0Rt	0	0Rt	0	0	0
	4-2	0	0Rt	0	0Rt	0	0Rt	0	0	0
	4-3	0	0Rt	0	0Rt	0	0Rt	0	0	0
	4-4	0	0	0	0Rt	0	0Rt	0	0	0
	4-5	0	0Rt	0	0Rt	0	0Rt	0	0	0
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	0	0	0	0	0	0	0	0	0
	5-2	0	0	0	0	0	0	0	0	0
	5-3	0	0	0	0	0	0	0	0	0
	5-4	0	0	0	0	0	0	0	0	0
	5-5	0	0	0	0	0	0	0	0	0

0=     No signs of systemic toxicity

Rt =     Light brown coloured residual test item on the ears

Table 6          Individual Bodyweights and Body weight Changes

Treatment Group	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle acetone/olive oil 4:1	1-1	19	20	1
	1-2	17	17	0
	1-3	20	18	-2
	1-4	21	20	-1
	1-5	19	20	1
Test Item 10% w/w in acetone/oliveoil 4:1	2-1	18	18	0
	2-2	19	20	1
	2-3	19	20	1
	2-4	18	19	1
	2-5	19	19	0
Test Item 25% w/w in acetone/oliveoil 4:1	3-1	18	18	0
	3-2	15	16	1
	3-3	19	19	0
	3-4	20	20	0
	3-5	19	19	0
Test Item 50% w/w in acetone/oliveoil 4:1	4-1	19	18	-1
	4-2	19	20	1
	4-3	17	18	1
	4-4	18	17	-1
	4-5	18	18	0
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	19	20	1
	5-2	18	18	0
	5-3	18	18	0
	5-4	18	19	1
	5-5	18	18	0

Applicant's summary and conclusion

Conclusions:

The substance was considered to be a sensitiser based on OECD TG 429.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. This study was conducted according OECD TG 429 and GLP principles. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of the test item as a solution in acetone/olive oil 4:1at concentrations of 50%, 25% or 10% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was performed with the known sensitiser,α‑Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1. Results showed the Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: the stimulation index was 16.10; 16.88; 9.15 and 10.63 for the concentrations 10%, 25%, 50% w/w in acetone/olive oil 4:1 and the positive control respectively. All results showed a positive result. At the 50% concentration light brown residues of the skin were seen, indicating that this % was possibly too high, though not noticed in the preliminary test. α-Hexylcinnamaldehyde, 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.The substance was considered to be a sensitiser under the conditions of the test, due to the absence of a dose-response the study receives Kl. 2.