1,4-Diazabicyclo[2.2.2]octane-2-methanol

Endpoint summary

Description of key information

skin sensitisation (OECD 429), LLNA: sensitising

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:

19 Mar - 14 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J (inbred, SPF-quality)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: Pre-screen test: 20 - 24 g; Main test: 18 - 23 g
- Housing: during experimental phase group housing in labeled Makrolon cages (MII type, height 14 cm) with a sheet of paper
- Diet: pelleted rodent diet (SM R/M-Z from Sniff Spezialdiäten GmbH, Soest, Germany)
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Mar 2014 To: 14 Apr 2014

Vehicle:

dimethylformamide

Concentration:

10, 25 and 50% (w/w)

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. Two test substance concentrations (25% and 50%) were tested.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.
No irritation and no signs of systemic toxicity were observed in any of the animals examined. White test substance remnants on the dorsal surface of the ears of both animals treated at 50% (between Days 1 and 4) did not hamper scoring for erythema. 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 50%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: If the results indicate a stimulation index (SI) ≥ 3, the test substance may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration on three consecutive days (Days 1, 2 and 3). The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
On Day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) and 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.
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 200 g for 10 minutes at 4 ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR).

Positive control substance(s):

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

Statistics:

The EC3 value was determined using linear interpolation.

Positive control results:

The results of a reliability test with three concentrations of hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v) was performed not more than 6 months previously (Oct 2013) using the same materials, animal supplier, animal strain and essential procedures. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.1 and 8.0 respectively. An EC3 value of 14.1% 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 11.9, 16.9, 14.4, 16.5, 14.5 and 13.4%.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.5, 2.3 and 3.9 respectively. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 35.9% was calculated.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 301, 449 and 749 DPM respectively. The mean DPM/animal value for the vehicle control group was 194 DPM.

Table1: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

group	TS [%]^1	animal	Size nodes^2		DPM^3/ animal	mean DPM ± SEM^4	mean SI ± SEM
			left	right
1	0	1	n	n	141	194 ± 22	1.0 ± 0.2
		2	n	n	206
		3	n	n	143
		4	n	n	248
		5	n	n	233
2	10	6	n	n	410	301 ± 38	1.5 ± 0.3
		7	n	n	212
		8	n	n	362
		9	n	n	298
		10	n	n	223
3	25	11	n	n	365	449 ± 52	2.3 ± 0.4
		12	n	n	593
		13	+	+	560
		14	n	n	366
		15	n	n	359
4	50	16	+	+	541	749 ± 72	3.9 ± 0.6
		17	+	+	750
		18	n	n	872
		19	n	n	649
		20	n	n	935

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

⁴ SEM = Standard Error of the Mean

Skin reactions: No irritation of the ears was observed in any of the animals examined. White test substance remnants on the dorsal surface of the ears of the animals treated at 50% (between Days 1 and 4) did not hamper scoring for erythema.

Systemic toxicity and body weights: 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.

Macroscopy of the auricular lymph nodes and surrounding area: The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal at 25% and two animals at 50%, which were enlarged. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:

other:

Remarks:

CLP/EU GHS Category 1B (H317) according to Regulation (EC) No 1272/2008

Conclusions:

CLP: Skin sens 1B, H317
DSD: Xi, R43

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD Guideline 429 and in compliance with GLP (Tosoh Corporation, 2014). Based on the results of a pre-screen test, test substance concentrations of 10%, 25% and 50% (w/w) in dimethyl formamide were selected for the treatment of mice in the main study. In this experiment, 5 female CBA/J mice per test group were treated with the test substance or vehicle dimethyl formamide alone for three consecutive days by open application on the ears (25 µL/ear). 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. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 301, 449 and 749 DPM respectively. The mean DPM/animal value for the vehicle control group was 194 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.5, 2.3 and 3.9 respectively. The data showed a dose-response and an EC3 value of 35.9% was calculated. No irritation of the ears was observed in any of the animals examined. The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal at 25% and two animals at 50%, which were enlarged. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The six-month reliability check with hexyl cinnamic aldehyde indicated that the LLNA is an appropriate model for testing for contact hypersensitivity.

In conclusion, since the test substance elicits an SI≥3, the substance is considered as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Justification for selection of respiratory sensitisation endpoint:
Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on skin sensitisation of the test substance meet the classification criteria for Skin Sens. 1B (H317) according to Regulation (EC) 1272/2008 and for Xi, R43 according to Directive 67/548/EEC.