Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 January 2016 to 2 March 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

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)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

EC Number:

267-032-7

EC Name:

Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

Cas Number:

67762-73-6

IUPAC Name:

Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): EXP1506833
- Substance type: UVCB
- Physical state: amber colored viscous liquid
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23g
- Housing: suspended solid floor polypropylene cages furnished with softwood flakes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10 %, 25 % and 50 % v/v in methyl ethyl ketone

No. of animals per dose:

4 animals per dose

Details on study design:

Groups of four mice were treated with the test item at concentrations of 50 %, 25 % or 10 % w/w in methyl ethyl ketone. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 four mice received the vehicle alone in the same manner.

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

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.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 labeled with the study number and dose concentration. The lymph node cell 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 pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5 % Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by ß-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 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 20 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:

None

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

44

Remarks on result:

other: Positive

Results 2

Parameter:

SI

Value:

1.22

Test group / Remarks:

10% v/v

Remarks on result:

other: Negative

Results 3

Parameter:

SI

Value:

1.41

Test group / Remarks:

25% v/v

Remarks on result:

other: Negative

Results 4

Parameter:

SI

Value:

3.5

Test group / Remarks:

50% v/v

Remarks on result:

other: Positive

Results 5

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle = 1708.28 dpm (213.54 dpm/Node) 10% w/w = 2091.34 dpm (261.42 dpm/Node) 25% w/w = 2408.25 dpm (301.03 dpm/Node) 50% w/w = 5977.14 dpm (741.14 dpm/Node)

Any other information on results incl. tables

Stimulation Index and results of each treatment

Concentration (%v/v) in methyl ethyl ketone	Stimulation Index	Result
10	1.22	Negative
25	1.41	Negative
50	3.50	Positive

Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (% w/w) in methyl ethyl ketone	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	0.21	0.22	0.24	0.22	0.20	0.20
overall mean (nm)		0.22		0.23		0.20
overall mean ear thickness change (%)		na		6.98		-6.98

Disintegrations per minute, disintegrations per minute/node and stimulation index

Concentration (% v/v) in methyl ethyl ketone	dpm	dpm/Node	Stimulation Index	Result
Vehicle	1708.28	213.54	na	na
10	2091.34	261.42	1.22	Negative
25	2408.25	301.03	1.41	Negative
50	5977.14	741.14	3.50	Positive

Individual Clinical Observations and Mortality Data

Concentration (% w/w) in methyl ethyl ketone	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	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
10	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
25	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
50	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0

Individual Body Weights and Body Weight Change

Concentration (% w/w) in methyl ethyl ketone	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	16.6	17.4	0.8
	1 -2	17.7	18.4	0.7
	1 -3	17.2	17.5	0.3
	1 -4	17.8	18.6	0.6
10	2 -1	18.2	18.8	0.6
	2 -2	19.2	19.8	0.6
	2 -3	18.1	18.6	0.5
	2 -4	17.7	18.1	0.4
25	3 -1	19.6	20.3	0.7
	3 -2	18.1	18.8	0.7
	3 -3	16.4	17.3	0.9
	3 -4	18.0	18.9	0.9
50	4 -1	20.1	20.7	0.6
	4 -2	18.3	19.2	0.9
	4 -3	20.5	20.9	0.4
	4 -4	19.0	20.3	1.3

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The Stimulation Index - expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group - provided a positive result for the highest suitable concentration tested. The test substance therefore is classified as sensitising according to the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The in vivo skin sensitisation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 429. A Local Lymph Node Assay (LLNA) was performed to assess the skin sensitization potential of the registered substance in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

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 four animals, were treated with 50 µL (25 µL per ear) of the registered substance as a suspension in methyl ethyl ketone at concentrations of 50 %, 25 % or 10 % w/w. A further group of four animals was treated with methyl ethyl ketone alone.

 

The Stimulation Index - expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group - provided a positive result for the highest concentration tested. The test substance therefore is classed as sensitising according to the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.