Reaction mass of 1,3-diacetoxypropan-2-yl 12-acetoxyoctadecanoate and 2,3-diacetoxypropyl 12-acetoxyoctadecanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-09-12 to 2007-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with no deviations and performed in accordance with GLP regulations

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-530-8

EC Name:

-

Cas Number:

736150-63-3

Molecular formula:

Not applicable

IUPAC Name:

1,3-bis(acetyloxy)propan-2-yl 12-(carboxyoxy)octadecanoate; 2,3-bis(acetyloxy)propyl 12-(acetyloxy)octadecanoate

Details on test material:

Name of test material (as cited in study report): TS-ED 532 (Batch D)
- Lot/batch No.: batch 4010534806
- Expiration date of the lot/batch: 2009-02-06

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: mouse (CBA/CaOlaHsd strain)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 19.5-19.9 g
- Housing: single in makrolon Type 1 , with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-71%
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

TS-ED 532 was used in concentrations of 10, 25, and 50 % (w/v).

TS-ED 532 was placed into a volumetric flask on a tared balance and the vehicle (acetone:olive oil (4+1) (v/v)) was quantitatively added. Based on the results from an initial solubility experiment the highest TS-ED 532 concentration was a 50%. The weight/volume dilutions were prepared by serial dilution. Homogeneity of TS-ED 532 in the vehicle was maintained during treatment with a magnetic stirrer. The preparations were made freshly before each dosing occasion.

No. of animals per dose:

Number of animals in test groups: 5
Number of animals in negative control group: 5
Number of animals in positive control group: 5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The data of a preliminary solubility experiment showed that the highest TS-ED 532 concentration that could be
technically achieved was a 50% solution.
- Irritation: NA
- Lymph node proliferation response: NA

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Mice were treated daily for three consecutive days with 25 μL of the appropriate concentration of TS-ED 532 on the dorsal surface of each ear (approximately 8 mm in diameter). Five days after the 1st topical application, all mice were administered 250 μL of 81.3 μCi/mL 3HTdR (approx. 20.3 μCi 3HTdR/mouse) by intravenous injection via a tail vein. Approximately 5 hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Ketamin/Xylazin/Midazolam.

The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5% trichloroacetic acid. The scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The ANOVA (Dunnett-test) was conducted to assess whether the difference between TS-ED 532 test groups and negative control (vehicle) group was statistically significant.

Statistical significance: p < 0.05.

Results and discussion

Positive control results:

Positive and vehicle controls were within the historical range ensuring the validity of the test. With the positive control an S.I. value of 5.42 was obtained at a concentration of 25% (See Table 1 below).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: SI: 1.00 (Control group). Si: 5.42 (Positive control group) SI: 2.19 (10 % (w/v)). SI: 1.99 (25 % (w/v)). SI: 2.20 ( 50 % (w/v)).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 1 below

Any other information on results incl. tables

The EC 3 value for TS-ED 532 could not be calculated, since all SI values were below 3.

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the mice and no systemic findings were observed during the study period. The body weights of the mice throughout the study were within the range commonly recorded for this strain and age.

Table 1 Calculations and results

TS-ED 532 concentration		Measurement DPM	Calculation	Result	Calculation	DPM per lymph node b)	Result
% (w/v)	Group		DPM-BG per animal (2 lymph nodes) a)	S.I.*	number of lymph nodes		S.I.
---	BG I	19	---	---	---	---	---
---	BG II	19	---	---	---	---	---
---	CG 1_1	1745	1726	---	---	---	---
---	CG 1_2	3523	3504	---	---	---	---
---	CG 1_3	2358	2339	---	---	---	---
---	CG 1_4	1550	1531	---	---	---	---
---	CG 1_5	2281	2262	---	10	1136.2	1.00
25	PG 1_1	6502	6483	2.9	---	---	---
25	PG 1_2	13972	13953	6.1	---	---	---
25	PG 1_3	14652	14633	6.4	---	---	---
25	PG 1_4	13704	13685	6.0	---	---	---
25	PG 1_5	12792	12773	5.6	10	6152.7^s	5.42
10	TG 1_1	4419	4400	1.9	---	---	---
10	TG 1_2	8958	8939	3.9	---	---	---
10	TG 1_3	4475	4456	2.0	---	---	---
10	TG 1_4	2353	2334	1.0	---	---	---
10	TG 1_5	4764	4745	2.1	10	2487.4	2.19
25	TG 2_1	2426	2407	1.1	---	---	---
25	TG 2_2	5273	5254	2.3	---	---	---
25	TG 2_3	5232	5213	2.3	---	---	---
25	TG 2_4	3736	3717	1.6	---	---	---
25	TG 2_5	6020	6001	2.6	10	2259.2	1.99
50	TG 3_1	4947	4928	2.2	---	---	---
50	TG 3_2	7548	7529	3.3	---	---	---
50	TG 3_3	3894	3875	1.7	---	---	---
50	TG 3_4	4660	4641	2.0	---	---	---
50	TG 3_5	4050	4031	1.8	10	2500.4	2.20

BG =  Background (1 ml 5% trichloroacetic acid) in duplicate

CG =  Control Group

PG =  Positive Control Group

TG=  Test Group

S.I. =  Stimulation Index

*    =  mean net DPM value per lymph node of the mouse divided by the mean net DPM value of the vehicle control group

^a)   =  The mean of the background value (BGI and BGII) was subtracted from the value.

^b)    =  DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a group by number of lymph nodes taken from that group

^S Mean DPM value for the group was according to the Dunnett test significantly higher than corresponding control value. The p value for the analysis was <0.05.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

TS-ED 532 was not a skin sensitiser in the LLNA study using concentrations of 10, 25 and 50%

Executive summary:

The dermal sensitization of TS-ED 532 was determined using the local lymph node assay in accordance with OECD Guideline for Testing of Chemicals 429. Groups of 5 female mice were dosed at: 0% (control), 10%, 25% and 50% solutions of TS-ED 532 diluted with a 4:1 mixture of acetone to olive oil.

No deaths occurred during the study period. No symptoms of local effects at the ears of the mice and no systemic findings were observed during the study period. The body weight of the mice throughout the study was within the range commonly recorded for this strain and age.

Stimulation indexes (SIs) for the 10% dose group ranged from 1.0 to 3.9 with a mean of 2.19. For the 25% dose group, the SIs ranged from 1.1 to 2.6 with a mean of 1.99. For the 50% dose group, the SIs ranged from 1.7 to 3.3 with a mean of 2.20. The SIs for the positive control ranged from 2.9 to 6.4, with a mean of 5.42. The EC3 value could not be calculated for TS-ED 532, since none of the tested concentrations induced an SI greater than 3.

None of the tested TS-ED 532 concentrations induced a statistically significant increase in DPM values when compared to the control. The positive control did cause a statistically significant increase which demonstrates the reliability and sensitivity of this assay to detect skin sensitization potential. The stimulation index is <3 which indicate that TS-ED 532 does not have the potential to cause skin sensitization.