Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July to 05 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol
EC Number:
204-104-9
EC Name:
Pentaerythritol
Cas Number:
115-77-5
Molecular formula:
C5H12O4
IUPAC Name:
pentaerythritol
Constituent 2
Reference substance name:
2,2-Bis(hydroxymethyl)-1,3-propanediol
IUPAC Name:
2,2-Bis(hydroxymethyl)-1,3-propanediol
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Pentaerythritol
- Physical state: crystalline solid
- Analytical purity: 98.8%
- Lot/batch No.: 4410102701
- Expiration date of the lot/batch: 18 December 2016
- Storage condition of test material: ambient temperature, protected from light
Specific details on test material used for the study:
- Name of test material (as cited in study report): Pentaerythritol
- Physical state: crystalline solid
- Analytical purity: 98.8%
- Lot/batch No.: 4410102701
- Expiration date of the lot/batch: 18 December 2016
- Storage condition of test material: ambient temperature, protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 18-22 g
- Housing: All mice were housed in pairs in solid-bottomed polycarbonate/polypropylene cages with a stainless steel grid top and an integrated food hopper. The control animals were housed remotely from the test animals. Sterilised white wood shavings were used as bedding. Cardboard play tunnels, nesting material and wooden chewsticks were placed in each cage as environmental enrichment.
- Diet: SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded, ad libitum
- Water: water from the public supply, ad libitum
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-21°C (target 19-23°C)
- Humidity (%): 49-74% (40-70%)
- Air changes (per hr): at least 10/hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle

IN-LIFE DATES: From: 12 August 2015 To: 21 August 2015

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: 25%
Main test: 0, 5, 10, 25%
No. of animals per dose:
Preliminary test: 2
Main test: 4
Details on study design:
Preliminary test: a preliminary test was conducted with 2 females, to investigate the toxicity/irritancy potential of the test material. The animals received an open application of 25 μL of the test item formulation onto the dorsum of each ear once daily for 3 consecutive days (Days 1 to 3). Animals were checked for viability early in the morning and again as late as possible on each day, and were examined daily for reaction to treatment. The body weight of each individual animal was recorded on Day 1 (before the first dose) and on Day 6. On Day 1 (predose) and Days 3 and 6 the thickness of both ears of each mouse was measured using digital callipers. In addition, both ears were observed for erythema and the scores recorded. Excessive local skin irritation is indicated by an erythema score ≥3 and/or increase in ear thickness of ≥25% on any day of measurement. A dose concentration giving rise to excessive local skin irritation is not suitable for administration on the main study. Animals were euthanised on Day 6. There were no signs of systemic toxicity, no clear effects on body weight, and no local irritation noted. The appropriate dose concentrations were selected for administration in the main study on the basis of these findings. Concentrations were taken from the concentration series detailed in the regulatory guidelines (OECD 429).

Main test: The concentrations selected from the main test were 0, 5, 10 and 25%. Animals received an open application of 25 μL of the appropriate formulation onto the dorsum of each ear on 3 consecutive days (Days 1 to 3). There was no treatment on Days 4 to 6. On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered saline (PBS) containing 21 μCi of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h after intravenous administration all animals were euthanised 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, a visual inspection of the nodes was made, 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 into conical centrifuge tubes. The lymph node cells were washed in an excess of PBS (approximately 1 mL) and the mesh was discarded. The lymph node cells were then centrifuged at approximately 1300 g for 10 min at 4°C. The supernatant was drawn off 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 2-8°C overnight. 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’, an aqueous-based solubiliser and the suspension transferred to a vial containing 10 mL scintillation fluid (Aquasafe 500 plus liquid). Incorporation of tritiated thymidine was measured by β-scintillation counting and was expressed as disintegrations per minute (DPM).

Calculation of results: 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 vehicle control group. The SI for the vehicle 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Group means and standard deviations were calculated for disintegrations per minute. Group means and standard deviations were calculated for body weights, along with body weight gains. The percentage of ear thickness of the preliminary test mice was calculated. Dixon’s Q-test (Dean and Dixon, 1951) for the detection of a single outlier was performed on disintegrations per minute values. No other formal statistical analyses were performed.

Results and discussion

Positive control results:
The experimental phase of a study using hexylcinnamaldehyde (HCA) at concentrations of 5% and 25% was completed in February 2015. The results from this study (Charles River Study No. 992477) produced stiumulation indices for HCA, when compared with the control group, of 1.7 and
10.5, for 5% and 25%, respectively. This was considered to provide evidence that the methodology used at the Test Facility is valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.7
Test group / Remarks:
5% test item
Parameter:
SI
Value:
1
Test group / Remarks:
10% test item
Parameter:
SI
Value:
0.9
Test group / Remarks:
25% test item
Parameter:
other: disintegrations per minute (DPM)
Value:
2 874
Test group / Remarks:
0% - vehicle control
Parameter:
other: disintegrations per minute (DPM)
Value:
1 935
Test group / Remarks:
5% test item
Parameter:
other: disintegrations per minute (DPM)
Value:
2 733
Test group / Remarks:
10% test item
Parameter:
other: disintegrations per minute (DPM)
Value:
2 580
Test group / Remarks:
25% test item

Any other information on results incl. tables

Preliminary test: there were no signs of systemic toxicity, no clear effects on body weight, and no local irritation noted.

Therefore, a 25% concentration of Pentaerythritol was selected as the highest concentration for the main study.

Main test: there were no systemic signs and no signs of local irritation in any animal during the observation period. Body weight gains were considered to be acceptable for mice of this age and strain.

Dixon’s Q-test for the detection of a single outlier was performed on the disintegrations per minute (DPM) data. Applying 95% confidence limits for a population of 4, none of the DPM values were found to be outliers.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, since treatment with Pentaerythritol at concentrations of up to 25% did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause skin irritation, and classification is not required.
Executive summary:

The delayed contact hypersensitivity potential of pentaerythritol was determined in a local lymph node assay (LLNA) according to OECD 429. The study was performed using female CBA/Ca mice. A formulation trial established that a 25% (250 mg/mL) concentration in acetone:olive oil (4:1 v/v) (AOO) was the maximum practicable concentration. A preliminary test was conducted by administering a 25% concentration of pentaerythritol to a group comprising 2 mice. As a result of the findings from the preliminary test, the following formulation concentrations were selected for the main study: 0, 5, 10 and 25%. The vehicle control group received AOO only. Treatment was for 3 consecutive days. Three days after the final application each animal received an intravenous injection of [methyl-³H] thymidine into the lateral tail vein. Approximately 5 hours later the draining lymph nodes were collected in order that incorporation of tritiated thymidine could be assessed by scintillation counting. There were no signs of systemic toxicity, and no signs of local irritation. The stimulation indices (SI) for 5%, 10% and 25%, when compared with the control group, were 0.7, 1.0 and 0.9, respectively. Under the conditions of the study, since treatment with pentaerythritol at concentrations of up to 25% did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause skin irritation. Pentaerythritol does not meet the criteria for classification as a skin sensitiser according to Regulation (EC) No 1272/2008.