Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 November to 2 December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study

Data source

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

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test material was di-pentaerythritol 93, batch no. 3810066489. One container holding approximately 700 g of test item was received under ambient conditions at Charles River on 04 September 2009. Upon arrival the test item, a white crystalline powder, was stored at ambient temperature, protected from light. A purity value of 94.93% and an expiry date of 03 September 2011 (ie 2 years from the date of despatch) were supplied by the Sponsor.
A read across is proposed from di-penta to pentaerythritol based on structural similarities between the substances.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
The animals were female (nulliparous and non-pregnant) mice of the CBA/Ca strain, supplied by Charles River, UK. They were 7 to 8 weeks old and
weighed 15.4 to 18.1 g on despatch. They were acclimatised for at least 8 days. On arrival, the animals were removed from their transport box in random order and were allocated to dose group by placing them in cages labelled with at least study number, animal number and group. Each animal received a subcutaneous implant which identified it individually within the study and which corresponded to that animal's number.
The animals were housed in groups of 2 or 3 in polycarbonate cages (dimensions 36.5 x 20.7 x 14 cm) with a stainless steel grid top and an integrated food hopper. Wood shavings were used as bedding and nesting material (‘Nestlets’) was provided. A wooden chewstick, supplied by Estap OÜ, Estonia, was placed in each cage as environmental enrichment. Analysis did not provide evidence of contamination that might have prejudiced the outcome of the study. Each cage was supplied with a water bottle.
From animal arrival to the end of the observation period, average daily environmental temperatures was approximately 21°C and the range for average daily relative humidity was approximately 44 to 62%. A 12 h light/dark cycle was in operation (light hours 0700 to 1900 h) with a minimum of 15 air changes per hour.
Rat and Mouse No. 1 Maintenance Diet (Special Diets Services, UK) and water taken from the public supply (Scottish Water, UK) were available ad libitum throughout the study. The results of diet and water analyses did not provide evidence of contamination and so the outcome of the study was not prejudiced.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Preliminary study: 50%
Main study: 10%, 25% and 50%
No. of animals per dose:
5 female mice per dose
Details on study design:
A preliminary study was conducted with 2 females and a 50% formulation concentration. There were no signs of systemic toxicity or local irritation and no effect on body weight was noted. Therefore, dose concentrations of 10%, 25% and 50% were selected as suitable non-toxic doses for administration in the main study.

Preliminary study: For 3 consecutive days (Days 1 to 3) animals received an open application of 25 μL of a 50% concentration of test item onto the dorsum of each ear. There was no further treatment. All animals were examined for reaction to treatment. 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 6. The animals were then discarded.

Main study: 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. All animals were examined for reaction to treatment. 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 the kill on Day 6 (the day of the thymidine injection).
On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered saline (PBS) containing 19.6 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. Owing to a procedural error, the lymph node collected from 2 animals in Groups 3 and from 2 animals in Group 4 cannot be attributed to specific individuals. The error has no effect on the group mean disintegrations per minute (DPM) values and
stimulation indices for Groups 3 and 4 and the Study Director’s interpretation of the study data is not affected.
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 8oC 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. 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.

Results and discussion

Positive control results:
The stimulation indices in a recent positive control study were 1.2, 1.5 and 5.0 for 5%, 10% and 25% hexylcinnamicaldehyde, respectively.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation indices for mice treated with the test item at concentrations of 10%, 25% or 50%, when compared with the control group, were 0.6, 1.2 and 0.7 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM values for mice treated with the test item at concentrations of 0%, 10%, 25% or 50% were 1780, 1086, 2051 and 1292, respectively.

Any other information on results incl. tables

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

Individual and Group Mean Scintillation Counts (DPM)

Treatment

Animal

DPM

Group Mean DPM

Stimulation Index

Control (vehicle only)

1

1033

1780

1

2

1852

3

1853

4

2598

5

1565

10% Di-Penta

6

1545

1086

0.6

7

1121

8

679

9

1893

10

191

25% Di-Penta

#

2392

2051

1.2

#

1601

13

2787

#

1331

#

2143

50% Di-Penta

#

2952

1292

0.7

#

575

18

466

#

565

#

1900

# The animal numbers from which these lymph nodes were collected were not documented correctly at the time of collection

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of the study, since treatment with Di-Pentaerythritol 93 at concentrations of up to 50% did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause sensitisation.
Executive summary:

This study investigated the delayed contact hypersensitivity potential of the test item, Di-Pentaerythritol 93 in a LLNA using CBA/Ca mice.

A preliminary test was conducted using 2 females. Each mouse received an open application of 25 μL of a 50% formulation of Di-Pentaerythritol 93 (Di-Penta 93) onto the dorsum of each ear on 3 consecutive days. There was no evidence of systemic toxicity or local irritation and, as a result of these findings, formulation concentrations were selected for the main study.

In the main study, three groups, each consisting of 5 females, were treated in the same manner as the preliminary test mice with concentrations prepared at 10%, 25% or 50%, respectively, also for 3 consecutive days. The vehicle was propylene glycol and one group of 5 females received only this and acted as controls. Three days after the final application each animal received an intravenous injection of [methyl-³H] thymidine into the lateral tail vein and 5 h later the draining lymph nodes were collected in order that incorporation of tritiated thymidine could be assessed by scintillation counting. There were no systemic signs noted in any animal during the observation period and body weight changes were considered to be acceptable for mice of this age and strain. The stimulation index (SI) values for the mice treated with Di-Penta 93 at concentrations of 10%, 25% or 50%, when compared with the control group, were 0.6, 1.2 and 0.7, respectively.

No evidence of skin sensitisation potential was seen under the conditions of this LLNA. The substance does therefore not require classification as a skin sensitiser under CLP.