Bis(6-methylalkyl-5-en-2-yl)dihydro-heteropolycycle

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb. - May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST SYSTEM

Species: Mouse, CBA strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Charles River France, L’Arbresle Cedex, France

Number of animals: 20 females (nulliparous and non-pregnant), five females per group.

Age and bodyweight: Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.

Identification: Tail mark with marker pen.

Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

ANIMAL HUSBANDRY

Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 20.6 - 23.2°C), a relative humidity of 30-70% (actual range: 35 - 63%) and 12 hours artificial
fluorescent light and 12 hours darkness per day.

Accommodation: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France).

Acclimatization period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment.

Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water: Free access to tap water.

Results of analysis for each batch of diet (nutrients) and results of quarterly analysis of diet (contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50 and 100%

No. of animals per dose:

5

Details on study design:

Preliminary irritation study:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest.
A series of two test substance concentrations was tested, selected from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The highest concentration, selected from this series, was the undiluted test substance.
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Main study:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

Allocation:
Group*1 animal numbers induction (% test substance)

1 01 - 05 0% (Acetone/Olive oil (4:1 v/v) )
2 06 - 10 25%
3 11 - 15 50%
4 16 - 20 100%

*1 five females each group

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 µl/ear) with the test substance
concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Treatment - Day 6:
All animals: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (GE Health care Buckinghamshire, UK). After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital (0.2 ml/animal Euthesate®; Sanofi Sante SV, Maassluis, The Netherlands). The draining (auricular) Iymph node of each ear was excised, The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 ml PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of Iymph node cells (LNC) was prepared in PBS by gentle separation
through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) at 4° C during the night.

Radioactivity measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 ml TCA and transferred to 10
ml of Ultima Gold cocktail (PerkinEImer Life and Analytical Sciences, Boston, MA, US) as the
scintillation fluid. Radioactive measurements were performed using a Packard scintillation
counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5
minutes whichever comes first. The Packard 2800TR was programmed to automatically subtract
background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:

Mortality/Viability: Twice daily.

Toxicity: At least once daily.

Body weights: On Days 1 (pre-treatment) and 6.

Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (Iocal) effects were recorded.

Grading Irritation Reactions:

Erythema and eschar formation:
No erythema ..................................................................................................................... 0
Slight erythema (barely perceptible) ............................................. ................................. 1
Well-defined erythema ..................................................................................................... 2
Severe erythema (beet redness) to slight eschar formation (injuries in depth) ...........3

Oedema formation:
No oedema ....................................................................................................................... 0
Slight oedema (barely perceptible) ................................................................................. 1
Moderate oedema............................................................................................................. 2
Severe oedema ................................................................................................................. 3

Positive control substance(s):

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

Statistics:

The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation.

Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A
comparison of statistical approaches to the derivation of EC3 values from locallymph node
assay dose responses. J Appl ToxicoI1999;19:261-266.

Results and discussion

Positive control results:

Reliability Check:

group treatment induction mean SI ± SEM
DPM ± SEM

2 experimental 5% test substance 249 ± 34 1.4 ± 0.3
3 experimental 10% test substance 393 ± 56 2.2 ± 0.3
4 experimental 25% test substance 1107 ± 243 6.1 ± 0.3

1 vehicle control Acetone : Olive oil (4:1) 183 ± 43 1.0
*. five females each group

group 2 | treatment experimental | induction 5% test substance | mean (DPM ± SEM) 249 ± 34 | SI ± SEM 1.4 ± 0.3;
group 3 | treatment experimental | induction 10% test substance | mean (DPM ± SEM) 393 ± 56 | SI ± SEM 2.2 ± 0.3;
group 4 | treatment experimental | induction 25% test substance | mean (DPM ± SEM) 1107 ± 243 | SI ± SEM 6.1 ± 0.3;

group 1 | treatment vehicle control | Acetone : Olive oil (4:1) | mean (DPM ± SEM) 183 ± 43 | SI ± SEM 1.0;

CONCLUSION
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 2.2 and 6.2 respectively. An EC3 value of 13.1% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 8.8, 5.5, 7.3 and 10.3%. Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

32.9

Results 2

Parameter:

SI

Value:

2.3

Variability:

SEM: ± 0.9

Test group / Remarks:

group 2: 25% test substance (Vehicle: Acetone/Olive oil (4:1 v/v))

Results 3

Parameter:

SI

Value:

4.4

Variability:

SEM: ± 1.7

Test group / Remarks:

group 3: 50% test substance (Vehicle: Acetone/Olive oil (4:1 v/v))

Results 4

Parameter:

SI

Value:

8.5

Variability:

SEM: ± 2.6

Test group / Remarks:

group 4: 100% test substance

Results 5

Parameter:

SI

Value:

1

Test group / Remarks:

group 1: 0% test substance; vehicle control (Vehicle: Acetone/Olive oil (4:1 v/v))

Results 6

Parameter:

other: Disintegration per minute (DPM)

Value:

710

Variability:

SEM: ± 204

Test group / Remarks:

group 2: 25% test substance (Vehicle: Acetone/Olive oil (4:1 v/v))

Results 7

Parameter:

other: Disintegration per minute (DPM)

Value:

1 371

Variability:

SEM: ± 353

Test group / Remarks:

group 3: 50% test substance (Vehicle: Acetone/Olive oil (4:1 v/v))

Results 8

Parameter:

other: Disintegration per minute (DPM)

Value:

2 669

Variability:

SEM: ± 255

Test group / Remarks:

group 4: 100% test substance

Results 9

Parameter:

other: Disintegration per minute

Value:

313

Variability:

SEM: ± 91

Test group / Remarks:

group 1: 0% test substance; vehicle control (Vehicle: Acetone/Olive oil (4:1 v/v) )

Cellular proliferation data / Observations:

Mean DPM/animal values for the experimental groups treated with test substance
concentrations 25, 50 and 100% were 710, 1371 and 2669 respectively.
The mean DPM/animal value for the vehicle contral group was 313.

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

 

Skin reactions after epidermal exposure and body weights

animal number	% test substance^1	Day 1		Day 3
		Body weight (g)		skin reactions dorsal surface ear				Body weight (g)
				left		right
				erythema	oedema	erythema	oedema
1	50	25		2	0	1	0	24
2	100	22		1	0	2	0	22

 

¹. Vehicle:Acetone/Olive oil (4:1 v/v) .

 

 

MAIN STUDY

Skin reactions, body weights and relative size auricular lymph nodes

group	% test substance^1	an^2		Day1		Day3					Day6
				bw (g)^3		skin reactions dorsal surface ear					bw (g)^3		size nodes^4
						left		right					left	right
						erythema	oedema	erythema	oedema
1	0%	1		22		0	0	0	0		22		n	n
	(vehicle)	2		24		0	0	0	0		22		n	n
		3		20		0	0	0	0		19		n	n
		4		21		0	0	0	0		22		n	n
		5		23		0	0	0	0		22		n	n
2	25%	6		21		1	0	1	0		20		n	n
		7		24		1	0	1	0		24		-	n
		8		24		1	0	1	0		25		n	n
		9		22		1	0	1	0		22		n	n
		10		24		1	0	1	0		23		n	n
3	50%	11		20		2	0	1	0		19		n	n
		12		21		1	0	2	0		20		n	n
		13		22		2	0	2	0		21		n	n
		14		24		1	0	1	0		23		n	n
		15		21		2	0	2	0		20		n	n
4	100%	16		21		2	0	2	0		21		n	+
		17		22		2	0	2	0		23		n	n
		18		21		2	0	2	0		21		+	+
		19		24		2	0	2	0		24		n	n
		20		23		2	0	2	0		23		++	+

¹.Vehicle:Acetone/Olive oil (4:1 v/v) .

². Animal number.

³.Body weight (grams).

⁴.Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

These results indicate that the test substance could elicit an SI >= 3. The data showed a dose-response and an EC3 value of 32.9% was calculated.

This conclusion should be taken with care, since a dose related increase in irritation of the ears was seen. It is known that irritation might stimulate the nodes, causing a false positive outcome of the local lymph node assay. Based on the laboratory experience, irritation might induce an SI up to approximately 4. The SI of 8.5 in this study was considered indicative for sensitisation.

Based on these results:
- according to the recommendations made in the test guidelines, Sa03 would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (New York and Geneva, 2003), Sa03 should be classified as skin sensitizer (Category 1).

- according to the ECETOC classification scheme for potency, Sa03 would be regarded as weak skin sensitizer.

- according to the CLP Regulation (CE) 1272/2008, Sa03 should be classified as Skin Sens. 1B.

Executive summary:

Assessment for Contact Hypersensitivity to Sa03 in the Mouse (Local Lymph Node Assay).

 

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2002),

EC, Council Directive 67/548/EEC, Annex V, B.42 (2004);

EPA, OPPTS 870.2600 (2003) “Skin Sensitisation”.

 

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, three groups of five experimental animals were treated with test substance concentrations of 25%, 50% or 100% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

A dose related increase in irritation of the ears was seen, from slight erythema in all animals at low dose up to well-defined erythema in all animals at high dose. No oedema was observed in any of the animals examined.

 

The majority of nodes were considered normal in size, except for the nodes of three animals treated at 100% which were enlarged and one node of one animal treated at 25% was reduced in size.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 710, 1371 and 2669 respectively.

The mean DPM/animal value for the vehicle control group was 313.

 

The SI values calculated for the substance concentrations 25, 50 and 100% were 2.3, 4.4 and 8.5 respectively.

 

These results indicate that the test substance could elicit an SI >=3. The data showed a dose-response and an EC3 value of 32.9% was calculated.

 

This conclusion should be taken with care, since a dose related increase in irritation of the ears was seen. It is known that irritation might stimulate the nodes, causing a false positive outcome of the local lymph node assay. Based on the laboratory experience, irritation might induce an SI up to approximately 4. The SI of 8.5 in this study was considered indicative for sensitisation. 

 

Based on these results:

- according to the recommendations made in the test guidelines, Sa03 would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (New York and Geneva, 2003), Sa03 should be classified as skin sensitizer (Category 1).

- according to the ECETOC classification scheme for potency, Sa03 would be regarded as weak skin sensitizer.

- according to the CLP Regulation (CE) 1272/2008, Sa03 should be classified as Skin Sens. 1B.