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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25. Oct. 2004 - 15. Nov. 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species : Mouse, CBA strain, inbred, SPF-Quality. Recognised 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 : 15 females (three groups of five females each group, nulliparous and non-pregnant).
Age and bodyweight : Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification : Tailmark.
Control animals : The results of the vehicle control animals in NOTOX Project 418534 were used as reference for the initially treated animals of this study. The vehicle control animals were treated using the same vehicle using the same procedures and within the same time frame as this study.
Reliability check : The results of a reliability test performed not more than 6 months previously or 2 months afterwards are summarised in the Appendix of the study report. Similar procedures were used in the reliability test and in this study.
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: 19.4 - 21.9°C), a relative humidity of 30-70% (actual range: 41 - 75%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation : Individual housing in labelled Macrolon cages (type I; height 12.5 cm) containing sterilised sawdust as bedding material (Woody-Clean type 3/4; 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 II type; height 15 cm). Paper (Enviro-dri, BMI, Helmond, The Netherlands) was supplied as cage-enrichment.
Diet : Free access to standard pelleted laboratory animal diet (code VRF 1, Altromin, Lage, Germany).
Water : Free access to tap-water.
Certificates of analysis for ingredients and/or contaminants of diet, sawdust, paper and water were examined and then retained in the NOTOX archives. Results of analysis were assessed and did not reveal any findings that were considered to have affected study integrity.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
100 %, 50 %, 25 %
No. of animals per dose:
5
Details on study design:
INDUCTION - Days 1, 2 and 3
Experimental animals : The dorsum surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time each day.
TREATMENT - Day 6:
All animals: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 pCi of 3H-methyl thymidine (Amersham Pharmacia 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 11m). 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 ml 5% trichloroacetic acid (TeA) at 4 °C for approximately 18 hours. Precipitates were recovered by centrifugation at 200g for 10 minutes, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold (Packard) as the scintillation fluid.
Radioactivity measurements : All radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert CPM to DPM.
Interpretation : 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 2 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM (NIH publication; No 99-4494, February 1999). The results were evaluated according to the OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998) and the EC criteria for classification and labelling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities). If possible, an EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation (Reference 2).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the ALPHAHEXYLCINNAMICALDEHYDE, TECH. 85% (CAS no. 101-86-0) concentrations 5, 10 and 25% were 1.0, 3.2 and 7.1 respectively. 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
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 1.1, 1.0 and 0.7 respectively. There was no indication that the test substance could elicit an SI > = 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 411, 385 and 243 respectively. The mean DPM/animal value for the vehicle control group was 373 (NOTOX Project 418534).

Any other information on results incl. tables

Calculation of Stimulation Index (SI)

Group

Treatment

Induction

Mean

SI±SD

 

 

 

DPM±SD

 

2

Experimental

25 % test substance

411±188

1.1±0.5

3

Experimental

50 % test substance

385±105

1.0±0.3

4

experimental

100 % test substance

243±137

0.7±0.6

1

Vehicle control

Dimethyl formamide

373±48

1.0

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The SI values calculated for the substance concentrations 25, 50 and 100% were 1.1, 1.0 and 0.7 respectively. There was no indication that the test substance could elicit an SI > = 3. 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), 1-ETHYL-3-METHYL IMIDAZOLIUm ETHYLSULFATE should not be regarded as a skin sensitiser.
Executive summary:

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2002), Paris Cedex; EC, Council Directive 67/548/EEC, Annex V, B.42 (2004); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600 "Skin Sensitisation" (2003). 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 25%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (N,N-dimethylformamide (NOTOX Project 418534). 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. After precipitating the DNA of the lymph node cells, radioactivity measurements were done. The majority of the nodes of the experimental and control groups were considered normal in size. The nodes of two animals treated at 25 and 100% were decreased in size. No other macroscopic abnormalities of the nodes were noted. Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 411, 385 and 243 respectively. The mean DPM/animal value for the vehicle control group was 373 (NOTOX Project 418534). The SI values calculated for the substance concentrations 25, 50 and 100% were 1.1, 1.0 and 0.7 respectively. There was no indication that the test substance could elicit an SI > = 3. 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), 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE should not be regarded as a skin sensitiser.

Based on these results and according to the:

- OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE does not have to be classified for sensitisation by skin contact.

- EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.