2,5-dimethyl-1H-pyrrole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline Study

Data source

Reference

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2,5-dimethyl-1H-pyrrole

EC Number:

210-913-8

EC Name:

2,5-dimethyl-1H-pyrrole

Cas Number:

625-84-3

Molecular formula:

C6H9N

IUPAC Name:

2,5-dimethyl-1H-pyrrole

Constituent 2

Reference substance name:

2,5-Dimethylpyrrole

IUPAC Name:

2,5-Dimethylpyrrole

Test material form:

other: yellowish liquid

Details on test material:

Purity 99.7%

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA strain

Sex:

female

Details on test animals and environmental conditions:

Mouse, CBA strain, inbred, SPF-Quality.
Recognized by the international guidelines as the recommended
test system (e.g. OECD, Ee, EPA).
Source: Charles River France, L'Arbresle Cedex, France
20 females (nulliparous and non-pregnant), five females per group.
Young adult animals (approx. 11-12 weeks old) were selected.
Body weight variation was within +/- 20% of the sex mean.
Tail mark with marker pen.
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.
The results of a reliability test with Hexylcinnamic aldehyde,
performed not more than 6 months previously or 2 months
afterwards, are summarized in the Appendix. Similar procedures
were used in the reliability test and in this study.
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: 19.1 23.0°
C), a relative humidity of 30-70% (actual range: 33 - 73%) 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 labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized
sawdust as bedding material (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren , The
Netherlands).
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 (Mill type; height 18 cm). Paper (Enviro-dri, TecniLab-BMI BV,
Someren, The Netherlands) was supplied as cage-enrichment.
Diet
Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage,
Germany)
Water
Free access to tap water.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

1% test substance
2,5% test substance
5% test substance

No. of animals per dose:

Three groups of five animals were treated with one test substance concentration per group.
One group of five animals was treated with vehicle.

Details on study design:

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 maximum
concentration that could technically be applied.
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). One animal was treated with the undiluted test substance and the other with 50%. Both
animals were sacrificed moribund on day 1.
Based on these results, four animals were treated with a 5, 2.5, 1 or 0.5% 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.
Based on the results, two additional animals were treated in a similar manner with a 25 and
10% concentration. Both animals were sacrificed moribund on day 1.
Induction - Days 1, 2 and 3
Experimental animals:
The dorsal surface of both ears was epidermally treated (25 pi/ear) with the test substance
concentration, at approximately the same time per 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.
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 pCi of 3H-methyl thymidine (Amersham
Biosciences, Buckinghamshire, UK).
After approximately five hours, all animals were killed by intra peritoneal injection with
pentobarbital (0.2 ml/animal Euthesate®; Sanofi Sante BY, Maassluis, 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 Ilm). LNC were washed twice with an excess of
PBS by centrifugation at 200g for 10 minutes at 4° C. 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 (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the
scintillation fluid. 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 Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

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

Results and discussion

Positive control results:

The six monthly reliability check with Hexylcinnamic aldehyde indicates that the Local Lymph
Node Assay as performed at NOTOX is an appropriate model for testing for contact
hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 1 and 2.5 were 11.2 and 14.7 respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 1, 2,5 and 5% were 2023, 2652 and 1170 respectively. The mean DPM/animal value for the vehicle control group was 181.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Conclusions:

These results indicate that the test substance could elicit an SI >=3. No reliable EC3 value could
be calculated.Based on these results according to the recommendations made in the test guidelines, 2,5-DIMETHYLPYRROLE
would be regarded as skin sensitizer.

Executive summary:

The SI values calculated for the substance concentrations 1 and 2.5 were 11.2 and 14.7

respectively.

No SI value was calculated for the 5% group, since the outcome of this group cannot be used

for interpretation due to the observation of significant systemic toxicity.

These results indicate that the test substance could elicit an SI >= 3. No reliable EC3 value could

be calculated.

The six monthly reliability check with Hexylcinnamic aldehyde indicates that the Local Lymph

Node Assay as performed at NOTOX is an appropriate model for testing for contact

hypersensitivity.

Based on these results:

- according to the recommendations made in the test guidelines, 2,5-DIMETHYLPYRROLE

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), 2,5-DIMETHYL.PYRROLE

should be classified as skin sensitizer (Category 1).

- according to the EC criteria for classification and labeling requirements for dangerous

substances and preparations (Council Directive 67/548/EEC), 2,5-DIMETHYLPYRROLE

should be labeled as: may cause sensitization by skin contact (R 43).