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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th May 2013 to 12th August 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to relevant guidelines and GLP compliant.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Sodium Isobutyrate Solution
IUPAC Name:
Sodium Isobutyrate Solution
Constituent 2
Reference substance name:
Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate.
IUPAC Name:
Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate.
Test material form:
other: Semi liquid
Details on test material:
- Name of test material (as cited in study report): Sodium Isobutyrate Solution.
- Physical state: Light yellow semi liquid
- Analytical purity: 86.5%
- Composition of test material: confidential information

- Lot/batch No.: W195_02
- Expiration date of the lot/batch: 17th October 2013
- Storage condition of test material: Controlled room temperature.
- Other: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Rj mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France.
- Age at study initiation: 8 weeks old.
- Weight at study initiation: 19.1 - 20.9 grams
- Housing: Mice were caged in groups and mice were provided with glass tunnel-tubes.
- Diet (e.g. ad libitum): Animals received ssniff SM Rat/Mouse - Breeding & Maintenance, 15mm, autoclavable Complete diet for rats/mice, ad libitum.
- Water (e.g. ad libitum): Animals received tap water from the municipal supply from 500mL bottles, ad libitum.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15-20 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light daily, from 6am to 6pm.

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:
other: Pluronic
Concentration:
Vehicle: 1%
Preliminary Irritation/Toxicity test: 100% and 50% (w/v)
Main Assay: 100%, 50% and 25% (w/v)
Negative control: 1% vehicle
Positive control: 25% (w/v) in 1% Pluronic.
No. of animals per dose:
5 groups of four animals each group in main assay.
Details on study design:
A preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using 2 animals per dose at concentrations of 100% and 50% (w/v) in 1% Pluronic. This test was performed in a similar manner to the main assay, except it was terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not conducted.
In this preliminary Irritation/Toxicity Test, all mice weer observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for eythema. Ear thickness measuremetns were also made using a thickness gauge on Day 1 (pre-dose) , Day 3 (approximatley 48 hours after the first dose) and day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after sacrifice of the test animals.
Based on the results of the preliminary test, 100% and 50% (w/v) were determiend to be acceptable for the main test and the and the dose groups for the main test were as outlined in Table 1. Test animals were dosed with 25uL of the appropriate formulation using a pipetter on the dorsal surface of each ear. Each animal was dosed once a day for 3 consecutive days (days 1, 2 and 3), with no treatment on days 4, 5 and 6.
On Day 6, each test animal was intravenously injected via the tail vein with 250uL of sterile phosphate buffered solution (PBS) containing approximately 20uCi of 3HTdR and once injected, the mice were left for approximately 5 hours before euthanisation.
The draiing auricular lymph nodes were excised by makng a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes and the nodes were then removed using forceps. The nodes of each test group were pooled and collected in separate Petri dishes containing a small amount of PBS (1-2 ml) to keep the nodes wet before processing.
A single cell suspension of pooled lymph node cells was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10mL) and the pooled lymph node cells were pelletd with a relative centrifugal force of 190xg for 10 minutes at 4°C. After centrifugation, supernatants were discarded and th pellets were gently resuspended and 10mL of PBS was added to the tubes. This washing step was repeated twice and for each group of pooled lymph nodes.
After the final washing step, supernatants were removed and pellets were gently agitates and resuspended and 3mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight incubtation of approximately 18 hours at 2 - 8°C, precipitates were centriguged and supernatants were removed. Pellets were resuspened in 1mL of 5% (w/v) TCA solution and dispersed using an ultrasonic bath. Samples were transferred into a sutable sized scintillation vial filled with 10mL of scintillation liquid and thoroughly mixed. The vials were loaded into ß-scintillation counter and 3HTdR incorporation was measured (10 minute measurement).
The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background levels were also measured in duplicates by adding 1mL of 5% (w/v) TCA solution into a scintillation vial filled with 10mL of scintillation liquid.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
For the positive control group, a significant lymphoproliferative response was observed (SI of 19.4).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation index values were 1.5, 1.9 and 0.5 at concentrations of 100, 50 and 25% (w/v) respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The Disintegrations per minute were 1607.5, 2028.5 and 970.5 at concentrations of 100, 50 and 25% (w/v) respectively.

Any other information on results incl. tables

No mortality or signs of systemic toxicity were observed during the study. Alopecia was observed for all animals in the 100% (w/v) dose group and for one animal in the 50% (w/v) dose group on Days 4 -6. There were no signs of inflammation at the treated site.

No treatment related effects were observed on body weight.

DPM, DPN and Stimulation Index Values for all groups:

Test Group Name

Measured DPM/group

DPM

Number of lymph nodes

DPN

Stimulation Index

Background (5% (w/v) TCA)

29.5

-

-

-

-

Negative (vehicle) control (1% Pluronic)

1080

1050.5

8

131.3

1.0

Sodium Isobutyate Solution 100% (w/v) in 1% Pluronic

1637

1607.5

8

200.9

1.5

Sodium Isobutyate Solution 50% (w/v) in 1% Pluronic

2058

2028.5

8

253.6

1.9

Sodium Isobutyate Solution 25% (w/v) in 1% Pluronic

1000

970.5

8

121.3

0.9

Positive control (25% (w/v) HCA in 1% Pluronic)

20411

20381.5

8

2547.7

19.4.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, Sodium Isobutyrate Solution, when tested in a suitable vehicle, did not show any sensitisation potential in the Local Lymph Node Assay.
Executive summary:

This study was conducted to determine the skin sensitisation potential of Sodium Isobutyrate solution following dermal exposure, in accordance with OECD Test Guideline 429. The study was conducted using female CBA/J Rj mice. An initial Preliminary Irritation/Toxicity test was performed using dose levels of 100% and 50% (w/v) in 1% Pluronic. Based on the observations recorded in this preliminary test, the 100% (w/v) was selected as the top dose for the main test.

In the main assay, twenty test mice were allocated to five groups of 4 animals each. Three groups received Sodium Isobutyrate solution (formulated in 1% Pluronic) at 100%, 50% and 25% (w/v) concentrations. One group was used as the negative control group and received the vehicle (1% Pluronic) while another group was used as the positive control group and received 25% (w/v) HCA, dissolved in 1% Pluronic.

The test item solutions were applied on the dorsal surface of the ears of the test animals (25uL/ear) for three consecutive days (Days 1, 2 and 3), with no treatment on Days 4, 5 and 6. On day 6, the cell proliferation in the local lymph nodes was measured by the incorporation of 3HTdR and the values used to calculate the stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. Alopecia was observed for all animals in the 100% (w/v) dose group and in one animal in the 50% (w/v) dose group.

The stimulation index values were determined to be 1.5, 1.9 and 0.5 at concentrations of 100, 50 and 25% (w/v) respectively.

The disintegrations per minute were 1607.5, 2028.5 and 970.5 at concentrations of 100, 50 and 25% (w/v) respectively.

Under the conditions of this study, Sodium Isobutyrate Solution, when tested in a suitable vehicle, did not show any potential for skin sensitisation in the Local Lymph Node Assay.

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