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Toxicological information

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:
July 2010 - November 2010.
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully Guideline- and GLP-compliant
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): "GREWE-DIAMIN"
- Chemical name: 4-amino-2-methyl-5-Pyrimidinemethanamide
- Trade name: Grewediamin
- Molecular formula: C6H10N4
- CAS No.: 95-02-3
- Batch No.: M-622-035-200
- Appearance: White to yellowish crystals
- Solubility: Soluble
- pH: 10.3
- Conditions of storage: Refrigerated, in the dark
- Stability at conditions of storage: Stable
- Expiry date: February 2011

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Species, strain: Mice, CBA/CaOlaHsd.
- Supplier: Harlan Italy, I-33049 San Pietro al Natisone (UD).
- Sex, specification: Females, healthy young adult nulliparous animals.
- Age of the animals: About 8 weeks at the first administration.
- Weight range of the animals at the first application: 17.3 - 22.8 g.
- Number of animals: 5 animals/group (including spare animals):
15 animals for 3 test substance groups,
5 animals for the negative control group,
5 animals for the positive control group.
- Spare animals: The 5th animal of each group served as spare animal. It was treated in the same way as the other animals of its group.
Since all animals survived and all animals received the correct amount of 3HTdR all spare-animals were taken for the examination of the results.
The number of animals/group was then 5.

- Hygiene: Optimal hygienic conditions.
- Room temperature: Average of 21.3 °C (continuous monitoring and recording).
- Relative humidity: Average of 63.0 % (continuous monitoring and recording).
- Light: Only artificial light from 6.00 a.m. to 6.00 p.m.
- Cages: Single caging. Makrolon cages type II, (22 cm x 16.5 cm ground area, 15 cm high).
- Feed: Maintenance diet for rats and mice R/M-H (item V1534-300), autoclavable, ad libitum.
Manufacturer: Ssniff Spezialdiäten GmbH, D-59494 Soest.
- Water: Tap water from Makrolon-bottles with stainless steel canules, ad libitum.
- Bedding material: Aspen wood chips, ABEDD®, LAB & VET Service GmbH, Hasnerstraße 84/6, 1160 Wien (item LTE E-001).
Germ reduction by autoclaving; Changed 1/week.
- Environmental Enrichment: Nesting material, same material and source as the bedding material, are offered once a week. A "mouse house" (red
polycarbonate shelter, 9.8 x 8.8 cm ground area, 5.5 cm high) is offered per cage. Germ reduction by autoclaving.
- Acclimatisation: 6 days.

Study design: in vivo (LLNA)

dimethyl sulphoxide
CAS No 67-68-5
Group A (low dose): 2.5% (w/w) solution of "Grewe-diamin" in DMSO
Group B (mid dose): 5% (w/w) solution of "Grewe-diamin" in DMSO
Group C (high dose): 11.4% (w/w) solution of "Grewe-diamin" in DMSO
No. of animals per dose:
Details on study design:
A range finding study was performed with two animals/concentration at the following
concentrations:11.4 % and 5 % (11.4 % is the maximum achievable concentration suitable
for application). In this study the animals were treated in the same manner as
intended in the main study with 25 µL test substance on the dorsum of each ear on three
consecutive days. Ear thickness and body weight were measured on Day 1 before the first
administration and on Day 4 about 24 hours after the last administration.
None of the animals showed overt systemic toxicity, excessive local skin irritation at the
application sites or an important increase in ear thickness in the range finding study.

Test design
The test substance was administered in 3 concentrations to the dorsal surfaces of the ears of
the animals of the test substance groups. In a manner identical to that of animals in the
treatment groups animals of one negative control group and one positive control group were
treated with DMF and HCA respectively. Each animal was treated for 3 consecutive days.
3 days after the last administration the proliferation of the lymphocytes of the draining lymph
nodes was measured by the determination of the amounts of incorporated 3HTdR.

Incorporation of 3H-methyl thymidine in vivo
5 days after the first topical administration, each animal received 20 µCi 3HTdR by slow
intravenous administration. The injection solution containing nominal 80 µCi/mL 3HTdR was
prepared by the dilution of 720 µL 3HTdR (amersham pharmacia biotech, Kat. Nr.:TRA
310, batch B340, specific activity 2.0 Ci/mmol, radioactive concentration 1 mCi/mL,
radiochemical purity 95.6%, thymine content 0.3%) with 8.28 mL PBS. Several minutes
prior to 3HTdR administration the animals were kept restrained in Plexiglas-tubes and the tail
veins were visualised by placing the tails in warm water. Then 250 µL of the injection solution
were intravenously administered to each animal.

Preparation of single cell suspensions and determination of incorporated 3H-methyl thymidine
Approximately 5 hours after 3HTdR injection all animals were sacrificed by carbon dioxide
asphyxiation and the draining auricular lymph nodes were rapidly excised. The lymph nodes
of each group were pooled in PBS. A single cell suspension (SCS) of lymph node cells (LNC)
was prepared by gentle mechanical disaggregation of the pooled lymph nodes through a
70 µm cell strainer. The SCSs were then transferred into centrifuge tubes and LNC were
pelleted by centrifugation (4°C, max. 200 g, 10 min). Afterwards supernatants were removed
by aspiration. Then the LNC were resuspended and washed twice with PBS. After the final
washing the supernatants were removed leaving just a small volume (<0.5 mL) and
macromolecules were precipitated by incubation with 5 % trichloroacetic acid (TCA) at 4°C
overnight. Each precipitate was pelleted by centrifugation (4 °C, max. 200 g, 10 min) and
resuspended in 1 mL TCA. This suspension was transferred into scintillation vials containing
10 mL scintillation cocktail (Packard Bioscience: Ultima Gold, Bestellnr. 6013329) and 3HTdR
incorporation was determined with a ß-scintillation counter (TriCarb 2200CA, Packard
Instrument Co., protocol 4: single label 3H, dpm, AEC-quenchcurve in Ultima Gold from
21 June 2002).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Application of 25% HCA in AOO resulted in an SI of 6.9.
This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Resultsopen allclose all
Key result
> 3
Test group / Remarks:
10% solution
Remarks on result:
other: see Remark
A substance is regarded as a sensitiser in the LLNA if the test substance induces a 3-fold or greater increase in 3HTdR incorporation into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes, as indicated by the SI, together with the consideration of dose response. group K (negative control): 1 group A (low dose): 2.8 group B (mid dose): 4.3 group C (high dose): 5.8 group P (positive control): 6.9
other: disintegrations per minute (DPM)
Remarks on result:
other: group K (negative control): 4452 group A (low dose): 12262 group B (mid dose): 19058 group C (high dose): 25925 group P (positive control): 30876
Key result
> 3
Test group / Remarks:
30% solution
> 3
Test group / Remarks:
1% solution

Applicant's summary and conclusion

Interpretation of results:
Migrated information R43, May cause sensitisation by skin contact
According to OECD-Guideline 429 and Regulation (EC) No 440/2008 method B.42., Skin Sensitisation: Local Lymph Node Assay, "Grewe-diamin" is
regarded as a sensitiser in the LLNA.
According to the results of this study and to Directive 2001/59/EC for classification, the test substance "Grewe-diamin" needs to be labelled with
"R43 May cause sensitisation by skin contact".
Executive summary:


The Local Lymph Node Assay was performed to evaluate a possible skin sensitising

potential of "Grewe-diamin" according to the OECD-Guideline 429, 24 April 2002 and the

Regulation (EC) No 440/2008 method B.42., 30 May 2008.


The test substance was solved in Dimethylsulfoxide (DMSO) and was administered to three

groups of 5 female CBA/Ca mice. Administration was performed epicutaneously to the dorsal

surface of both ears, once a day on three consecutive days. The volume administered was

25 µL per ear.

Concentrations used:

Group A (low dose): 2.5% (w/w) solution of "Grewe-diamin" in DMSO

Group B (mid dose): 5% (w/w) solution of "Grewe-diamin" in DMSO

Group C (high dose): 11.4% (w/w) solution of "Grewe-diamin" in DMSO

Two groups with 5 animals each served as positive and negative controls. Both control

substances were administered under identical conditions as the test substances.

The following solutions served as control substances:

Group P (positive control): 25% (v/v) solution of hexyl cinnamic aldehyde (technical

grade, 85%) in acetone:olive oil (4:1, v/v)

Group K (negative control): DMSO

5 days after the first topical application, 3H-methyl thymidine was intravenously administered

to all mice via a tail vein. Approximately 5 hours later all animals were sacrificed, the draining

auricular lymph nodes were excised, pooled for each group, and single cell suspensions

were prepared. Then incorporation of 3H-methyl thymidine into the cells was determined

(liquid scintillation counter) and compared with the negative controls. The stimulation index

(SI) was calculated as the ratio of the disintegrations per minute (dpm) of the dosed groups

or of the positive control group to the dpm of the negative control group.



All animals survived till the end of the study. No adverse effects were noted in any animal.

Body masses and body mass gains were in the range to be expected from animals of the

same strain, sex and age.

No skin irritating effects at the application sites were observed in the test substance groups

and both control groups throughout the whole study.

Test substance remnants at the application sites of all animals of the positive control group

were observed on Days 2-6.

3H-methyl thymidine incorporation, stimulation indices

The calculated stimulation indices (test substance/negative control ratio) were decisive for

the grading of the potential of sensitisation: According to the guidelines the decision process

with regard to a positive response includes a stimulation index of equal to or greater than 3,

together with consideration of dose-response.

The SIs of the tested test substance concentrations were 2.8 (low dose), 4.3 (mid dose) and

5.8 (high dose).

Positive control

The positive control substance led to a stimulation index of 6.9, thus demonstrating the

validity of the experiment.


According to OECD-Guideline 429 and Regulation (EC) No 440/2008 method B.42., Skin

Sensitisation: Local Lymph Node Assay, "Grewe-diamin" is regarded as a sensitiser in the


According to the results of this study and to Directive 2001/59/EC for classification, the test

substance "Grewe-diamin" needs to be labelled with "R43 May cause sensitisation by skin contact".