[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FromDecember 6, 2011 to July 27, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to the OECD Guideline 429, with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: From 20 to 23 g bw.
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0ºC
- Humidity (%): 40-70%
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: December 7, 2011 To: January 9, 2012

Vehicle:

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

Concentration:

1 0 (Acetone/olive oil (4:1 v/v))
2 25
3 50

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Irritation: A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied. Two test substance concentrations were tested; a 10% and 25% concentration. Since severe skin effects were suspected at high concentrations the highest concentration was 25%.

The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

- Lymph node proliferation response: Not determined in the pre-screen test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures. The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6
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 (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph 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 lymph 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 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer 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 came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Positive control substance(s):

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

Positive control results:

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 2.0 and 7.4 respectively. An EC3 value of 12.8% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%.

Based on the results, it was concluded that the Local Lymph Node Assay is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Value:

1.8

Test group / Remarks:

25% test item

Parameter:

SI

Value:

4.3

Test group / Remarks:

50% test item

Parameter:

SI

Value:

9

Test group / Remarks:

100% test item

Parameter:

EC3

Value:

37

Table 1: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

 

Group	TS^1%	animal	Size nodes		DPM^3/animal	mean DPM±SEM^4	mean SI±SEM
			left	right
1	0	1	n	n	252	492±97	1.0±0.3
		2	n	n	267
		3	n	n	711
		4	n	n	639
		5	n	n	589
2	25	6	n	n	799	868±178	1.8±0.5
		7	n	n	380
		8	n	n	1345
		9	n	n	623
		10	n	n	1193
3	50	11	+	+	1905	2119±392	4.3±1.2
		12	+	+	1735
		13	+	+	2467
		14	+	+	3411
		15	+	+	1078
4	100	16	+	+	3932	4407±324	9.0±1.9
		17	+	+	3911
		18	+	+	4151
		19	+	+	5658
		20	+	+	4385

¹. TS = test substance (% w/w).

². Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be

normal).

³. DPM = Disintegrations per minute

⁴SEM = Standard Error of the Mean

 

 

 

PRE-SCREEN TEST:

Very slight erythema was noted in the animals at 10, 25, 50 and 100% during the observation period. Brown staining by the test substance prevented scoring for erythema in animals at 100% on Days 1, 2 and 3 after dosing. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

MAIN STUDY:

The slight irritation of the ears as shown by all animals treated at 50 and 100% was considered not to have a toxicologically significant effect on the activity of the nodes. Brown staining by the test substance prevented scoring for erythema in animals at 100% on Days 1, 2 and 3.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

All auricular lymph nodes of the animals at 50 and 100% considered larger in size as compared to the control group.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Condensation products of cyclopentanone and pentaldehyde, fractionation pitch was determined to be skin sensitizer.

Executive summary:

Assessment of Contact Hypersensitivity to Condensation products of cyclopentanone and pentaldehyde, fractionation pitch in the Mouse (Local Lymph Node Assay) was determined according to the 429 OECD and EU B42 Guidelines, with GLP.

Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w 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 with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

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.

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.8, 4.3 and 9.0 respectively.

These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 37% was calculated.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay is an appropriate model for testing for contact hypersensitivity.

Based on these results:

- according to the recommendations made in the test guidelines, Condensation products of cyclopentanone and pentaldehyde, fractionation pitch would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), Condensation products of cyclopentanone and pentaldehyde, fractionation pitch should be classified as skin sensitizer (Category 1B).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Condensation products of cyclopentanone and pentaldehyde, fractionation pitch should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study: Experimental result on test item Condensation products of cyclopentanone and pentaldehyde, fractionation pitch.

The possible skin sensitization potential of the test substance was carried out according to the 429 OECD Guideline (GLP study), by a LLNA test.

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.8, 4.3 and 9.0 respectively. These results indicate that the test substance could elicit an SI ≥ 3 therefore the product is considered to be a skin sensitizer. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 37% was calculated.

Migrated from Short description of key information:
Key study: OECD Guideline 429. GLP study. The test subtance was determined to be skin sensitizer.

Justification for selection of skin sensitisation endpoint:
Only one study available. GLP compliant and of high quality (Klimisch 1).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on these results:

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

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), the test substance should be classified as skin sensitizer (Category 1B).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, the test substance should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.