Registration Dossier

Administrative data

Description of key information

A local lymph node assay is available for the submission substance dimethylolpropane tech.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 September 2009 to 12 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Supplier: Charles River, UK limited
Age on despatch: 7-8 weeks
Weight on despatch: 16.1 to 19.6 g
Acclimatisation period: At least 8 days
Allocation of groups: Animals randomly removed from the transport box and allocating to cages by placing in cages
Housing: Polycarbonate cages witstainless steel grid tops with wood shavings and nesting material. A wooden chewstick was included. Two or three animals in each cage.
Humidity: 37-75%
Temperature: Approximately 21°C
Photoperiod: 12 hour light/dark cycle
Air changes: 15 changes/hour
Diet: Rat and Mouse No. 1 Maintenance Diet (Special Diet Services, UK); ad libitum
Water: Tap water (Scottish Water, UK); ad libitum
Vehicle:
dimethylformamide
Concentration:
25, 50 and 100%
No. of animals per dose:
Five
Details on study design:
In order to establish appropriate formulation concentrations for use in the main study, a
preliminary test was conducted using undiluted DMP tech. For 3 consecutive days (Days 1 to 3) two animals received an open application of 25 μL of undiluted test item onto the dorsum of each ear There were no signs of systemic toxicity or local irritation and no effect on body weight was noted. Therefore, dose concentrations of 25%, 50% and 100% were selected as suitable non-toxic doses for administration in the main study. These concentrations are taken from the concentration series detailed in the regulatory guideline (OECD Guideline No. 429).

For 3 consecutive days (Days 1 to 3) animals received an open application of 25 μL of the appropriate formulation onto the dorsum of each ear. There was no treatment on Days 4 and 5. On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered
saline (PBS) containing approximately 19 μCi of [methyl-3H] thymidine into the lateral tail vein.
Approximately 5 h after intravenous administration all animals were killed by exposure to a rising concentration of carbon dioxide and the major blood vessels were severed to exsanguinate. Each pair of draining auricular lymph nodes was collected from each animal and the animal was then discarded. A single cell suspension of lymph node cells from each paired sample was prepared by gentle disaggregation through a 200 μm mesh stainless steel gauze. The lymph nodes cells were washed in excess of PBS (approximately 1 mL) and then centrifuged at approximately 1300 g for 10 min at 4°C. The supernatant was drawn off and the mesh discarded and the pellet was washed a second time with approximately 1 mL PBS and then centrifuged (also at approximately 1300 g for 10 min at 4°C). Following centrifugation, the supernatant was discarded and the pellet was precipitated with approximately 1 mL 5% trichloroacetic acid at 2 to 8°C for approximately 21 h. The pellet was again centrifuged at approximately 1300 g for 10 min at 4°C and the supernatant discarded. The pellet was then re-suspended in 200 μL ‘Solvable’ and the suspension transferred to a vial containing 10 mL scintillation fluid. Incorporation of tritiated thymidine was measured by β-scintillation counting and was expressed as disintegrations per minute (DPM).

All animals were checked for viability early in the morning and again as late as possible on each day.

All animals were examined for reaction to treatment.

In the preliminary experiment, observations were conducted frequently on each day of dosing (predose, immediately post dose and approximately 1 and 2 h after dosing) and once daily thereafter until the kill, by cervical dislocation, on Day 7. The animals were then discarded.
In the main experiment, on each day of dosing the observations were conducted predose, immediately post dose and approximately 1 and 2 h post dose. Thereafter animals were observed once daily until kill on Day 6 (the day of the thymidine injection). The body weight of each individual animal was recorded on Day 1 (before the first dose) and on Day 6.

Results were corrected for background radiation and expressed as the Stimulation Index (SI). This was obtained by dividing the mean DPM obtained from each group by the mean DPM for the control group. The SI for the control group, therefore, is one. A positive response is indicated by an SI ≥3, together with consideration of dose-response and, where appropriate, statistical significance. As there was no SI value ≥3 recorded for any group, it was not possible to calculate the estimated concentration of test item that would produce a 3-fold increase in draining lymph node cell proliferation (the EC3 value).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No formal statistical analysis was carried out.
Positive control results:
The stimulation indices in a recent positive control study were 1.7, 2.4 and 5.0 for 5%, 10%
and 25% hexylcinnamicaldehyde, respectively.
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
100%
Key result
Parameter:
EC3
Remarks on result:
not determinable

No systemic signs were noted in any animal during the observation period. Body weight gains were considered to be acceptable for mice of this age and strain. The stimulation indices in a recent positive control study were 1.7, 2.4 and 5.0 for 5%, 10% and 25% hexylcinnamicaldehyde, respectively.

Individual and group mean scintillation counts

 

Dose group

DPM value

Mean DPM

Stimulation Index

Negative control

3720

3136

1

2595

2755

3697

2911

25% DMP Tech

3086

2763

0.9

3375

495

2607

4254

50% DMP Tech

3367

2642

0.8

1394

1891

2210

4349

100% DMP Tech

6835

5471

1.7

5531

7050

902*

2468

* Excluded from calculation of the mean due to spillage
DPM: disintegrations per minute

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, since treatment with Dimethylolpropane tech at concentrations of up to 100% (ie undiluted Dimethylolpropane tech) did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause skin sensitisation.
Executive summary:

A local lymph node assay was conducted with dimethylolpropane tech according to OECD guideline 429. The test article was treated at concentrations of 25, 50 and 100%, diluted where appropriate in dimethylformamide. The test article was applied (25 µL) onto the dorsum of each ear on Days 1, 2 and 3. On Day 6, an intravenous dose of phosphate buffereed saline was applied containing approximately 19 μCi of [methyl-3H] thymidine into the lateral tail vein. A single cell suspension of local lymph node cells was prepared from the auricular lymph nodes for each animal. The incorporation of tritiated thymidine was measured by β-scintillation counting and was expressed as disintegrations per minute (DPM). The stimulation index for the test article did not exceed 3, indicating that the test article does not have the potential to cause sensitisation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

A local lymph node assay was conducted with dimethylolpropane tech according to OECD guideline 429. The test article was treated at concentrations of 25, 50 and 100%, diluted where appropriate in dimethylformamide. The test article was applied (25 µL) onto the dorsum of each ear on Days 1, 2 and 3. On Day 6, an intravenous dose of phosphate buffereed saline was applied containing approximately 19 μCi of [methyl-3H] thymidine into the lateral tail vein. A single cell suspension of local lymph node cells was prepared from the auricular lymph nodes for each animal. The incorporation of tritiated thymidine was measured by β-scintillation counting and was expressed as disintegrations per minute (DPM). The stimulation index for the test article did not exceed 3, indicating that the test article does not have the potential to cause sensitisation.

Justification for classification or non-classification

The results of a local lymph node assay (LLNA) performed with Dimethylolpropane tech indicate a lack of skin sensitisation potential. No classification for skin sensitisation is therefore proposed according to the CLP Regulation.