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Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
Batch number 19902-1
Expiry data 31.10.2006

In vivo test system

Test animals

Details on test animals and environmental 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 k 3.0•‹C (actual range: 19.8 -22.3"C), a relative humidity of 30-70% (actual range: 32 - 56%) and 12 hours artificial
fluorescent light and 12 hours darkness per day.
Individual housing in labeled Macrolon cages (type I; height 12.5 cm) containing sterilised sawdust as bedding material woody-Clean type 314; Tecnilab-BMI BV, Someren , The Netherlands).
Acclimatisation period
The acclimatisation 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 polycarbonate cages (Macrolon I1 type; height 15 cm). Paper (Envirodri, BMI, Helmond, The Netherlands) was supplied as cage-enrichment.
Free access to standard pelleted laboratory animal diet (code VRF 1, Altromin, Lage, Germany).
Free access to tap water.

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
25 microliters/ear on both ears of 2.5-5.0-10.0 % w/w test material for the induction phase.
No. of animals per dose:
Details on study design:
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.
Three groups of five animals were treated with three test substance concentrations respectively. One group of five animals was treated with vehicle.
INDUCTION - Days 1,2 and 3
Experimental animals: The dorsum surface of both ears was epidermally treated (25 ~l learw) ith the test substance concentration, at approximately the same time each day.
Vehicle control animals: The control animals were treated the same as the experimental animals, except that, instead of the test substance. the vehicle alone was administered.
All animals:
Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 pCi of 3~-methythl ymidine (AmershamPharrnacia Biotech, NOTOX Substance 105624).
After approximately five hours, all animals were killed by intra peritoneal injection with an overdose of pentobarbital. 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 were recorded. The nodes were pooled for each animal in 3 ml PBS.
Tissue processing for radioactivity
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 pm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4' C. The DNA was precipitated with 3 ml5%
trichloroacetic acid (TCA) at 4O C for approximately 18 hours. Precipitates were recovered by centrifugation at 2009 for 10 minutes, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold (Packard) as the scintillation fluid.
All radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precision of k 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Average of 5 animals was determined with standard deviation.
The level of significance was p<0,05.

Results and discussion

Positive control results:
5% SI=1.0±0.4
10% SI=3.2±0.4
25% SI=7.1±0.4
An EC3 value of 9.5% 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.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: Ranges from 4.8 to 20.8
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Group treatment induction mean Mean DPM±SD SI±SD 2 experimental 2.5% test substance 2450 ± 740 4.8± 0.5 3 experimental 5% test substance 7969 ± 4406 15.6± 0.6 4 experimental 10% test substance 10609 ± 958 20.8 ± 0.7 1 vehicle control AcetonelOlive oil (4:l vlv) 511 ± 174 1 .O

Any other information on results incl. tables

No reliable EC3 value could be calculated.

Applicant's summary and conclusion

Interpretation of results:
Migrated information SI>3 Criteria used for interpretation of results: EU
The SI values calculated for the substance concentrations 2.5, 5 and 10% were 4.8, 15.6 and 20.8 respectively.
These results indicate that the test substance could elicit an SI ≥ 3. No reliable EC3 value could be calculated.
Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), N42-[2-(dimethylamine)ethoxy]ethyl}-Nmethyl-l,3-propanediamine would be regarded as skin sensitizer.
Executive summary:

Substance is skin sensitizer based on the results of this local lymph node assay.

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 epidermally exposed to a 2.5%, 5%, and 10%

concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but

with vehicle alone (AcetonelOlive oil (4: 1 vlv)).

Three days after the last exposure, all animals were injected with 3~-methythl ymidine and after five hours the

draining (auricular) lymph nodes were exercised.

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).

All nodes of the animals treated at 5% and the majority of nodes of the animals treated at 10% were enlarged in


Mean DPMIanimal values for the experimental groups treated with test substance concentrations 2.5, 5 and 10%

were 2450, 7969 and 10609 respectively.

The mean DPMIanimal value for the vehicle control group was 5 1 1.

The SI values calculated for the substance concentrations 2.5, 5 and 10% were 4.8, 15.6 and 20.8 respectively.

These results indicate that the test substance could elicit an SI 2 3. No reliable EC3 value could be calculated.

The six monthly reliability check with Hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as

performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Based on these results and according to the recommendations made in the test guidelines (OECD No. 429, EC

B.42 and EPA OPFTS 870.2600), N-12-[2-(Dimethylamine)Ethoxy]Ethyl)-N-Meth1y,l3-- Propanediamine

would be regarded as skin sensitizer.