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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 January to 24 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully Guideline- and GLP-compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
Details on test material:
- Name of test material (as cited in study report): "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde"
- Molecular formula (if other than submission substance): C5H10O3
- Physical state: Colourless, high viscous
- Composition of test material, percentage of components: ~ 82 % Dimethylpropanale, ~ 10 % Hydroxyethanesulfonic acid, Na-salt (CAS No.: 870-72-4), ~ 6 % water, ~ 2 % Potassiumformiate (CAS No.: 590-29-4)
- Lot/batch No.: CH 138067/001
- Expiration date of the lot/batch: 30 November 2010
- Stability under test conditions: Min. 1 year
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- 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: 17.0 - 22.9 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.

ENVIRONMENTAL CONDITIONS
- Hygiene: Optimal hygienic conditions.
- Room temperature: Average of 21.2°C (continuous monitoring and recording, shortly interrupted due to calibration of the data recorder).
- Relative humidity: Average of 58.9 % (continuous monitoring and recording, shortly interrupted due to calibration of the data recorder).
- 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, Dominik Mayr KEG, A-8580 Köflach.
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: 13 days.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
CAS No 67-68-5
Concentration:
•Group A (low dose): 2.5% (w/w) in DMSO
•Group B (mid dose): 5% (w/w) in DMSO
•Group C (high dose): 11.9% (w/w) in DMSO
•Group P (positive control):     25% (v/v)solution of hexyl cinnamic aldehyde (technical grade, 85%) in acetone:olive oil (4:1, v/v)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A range finding study was performed with two animals/concentration at the following concentrations: 11.9% and 5% (11.9% is the maximum achievable concentration suitable for application, see above). 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.
Therefore 11.9% was chosen as the highest test substance concentration and ear thickness measurement was not performed in the main study.
As mid concentration 5% was chosen, since 11.9% is very near at the proposed 10%.


MAIN STUDY
Bias control
The individual animals were allocated to their groups by random numbers.

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 DMSO 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 1.296 mL 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 14.904 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 beta-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:
On Days 3 and 4 all animals of the positive control group had slight erythema on the application sites indicating slight irritative skin reactions.
Application of 25% HCA in AOO resulted in an SI of 5.6.
This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
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. group K (negative control): 1 group A (low dose): 0.9 group B (mid dose): 1.1 group C (high dose): 0.9 group P (positive control): 5.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: group K (negative control): 6124 group A (low dose): 5213 group B (mid dose): 6745 group C (high dose): 5217 group P (positive control): 34509

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
"3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" is regarded as a non sensitiser in the LLNA according to OECD-Guideline 429 and Regulation (EC) No 440/2008 method B.42., Skin Sensitisation: Local Lymph Node Assay, since the SIs of all examined test substance concentrations were clearly smaller than 3.

According to the results of this study and to Directive 2001/59/EC for classification, the test substance "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" needs not 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 "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" ("Novaltan PF") according to the OECD-Guideline 429,and theRegulation (EC) No 440/2008 method B.42.,30 May 2008.

 

 

Method

 

The test substance was solved in Dimethylsulfoxide (DMSO) and was administered to three groups of 4 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 in DMSO

·      Group B (mid dose):       5% (w/w) solution of in DMSO

·      Group C (high dose):      11.9% (w/w) solution of in DMSO

 

Two groups with 4 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 of3H-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.

Results

 

General

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 the negative control group throughout the whole study. Slight erythema was noted in all animals of the positive control group on Days 3-4, indicating slight local skin irritation.

 

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 0.9 (low dose), 1.1 (mid dose) and 0.9 (high dose).

 

Positive control

The positive control substance led to a stimulation index of 5.6, thus demonstrating the validity of the experiment.

Conclusion

According to OECD-Guideline 429 and Regulation (EC) No 440/2008 method B.42.,Skin Sensitisation: Local Lymph Node Assay, "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" is regarded as a non sensitiser in the LLNA.

 

According to the results of this study and to Directive 2001/59/ECfor classification, the test substance "3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde" needs not to be labelled with "R43 May cause sensitisation by skin contact".