propylidynetrimethanol, ethoxylated, esters with acrylic acid and its reactions products with dibutylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

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:

propylidynetrimethanol, ethoxylated, esters with acrylic acid and its reactions products with dibutylamine

EC Number:

701-363-4

Molecular formula:

Not specified UVCB - Reaction product of ethoxylated (0-7 EO) propylidenetrimethanol with acrylic acid and dibutylamine (propylidenetrimethanol with acrylic acid : dibutylamine = 5:1)

IUPAC Name:

propylidynetrimethanol, ethoxylated, esters with acrylic acid and its reactions products with dibutylamine

Details on test material:

- Name of test material (as cited in study report): Laromer LR 8869
- Physical state: colorless, clear liquid
- Analytical purity: for details see analytical report no. 11L00263
- Purity test date: 2012-01-11
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 2012-07-4
- Stability under test conditions: not indicated by sponsor
- Storage condition of test material: at room temperature, light protected, avoid temperatures above 25°C

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl (1st prestest and main study); CBA/CaOlaHsd (2nd and 3rd pre-test)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V. (2nd and 3rd pre-test); Charles River UK (1st pre-test and main study)
- Age at study initiation: 9-10 weeks (main study); 8-10 weeks (pre-tests)
- Weight at study initiation: 17.1 - 20.5g
- Housing: group in Makrolon Type II / III with wire mesh top
- Diet (e.g. ad libitum): Pelleted standard diet, ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-100% (acclimatisation); 45-65% (main study)
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

planned
0.5%, 1%, 2.5% (no signs of excessive local skin irritation were observed at 1% and 2.5% during a pre-test)
as verified by analysis:
0.5%, 0.6%, 2.5%

No. of animals per dose:

2 (pre-test)
5 (main study)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 100%
- Irritation: at 10%, 25%, 50%, 100% irritation and an increase in ear thickness was observed. Animals treated with 100% of the test substance were sacrificed on day 3 due to distinctly reduced spontaneous activity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with 25µl/ear/day, was spread over the entire dorsal surface (app. 8 mm in diameter) of each ear once daily for three consecutive days. Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline (PBS) containing 20.4 μCi of 3HTdR (equivalent to approximately 81.5 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Criteria used to consider a positive response:
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:
The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1 v/v) was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Positive control substance(s):

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

Statistics:

One-Way-ANOVA. In case of significant results, multiple comparisons were performed with the Dunnett test and the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers

Results and discussion

Positive control results:

The periodic positive control was performed in August 2011
SI 5% = 1.35; 10% = 2.18; 25% = 8.08
The results are within the historical control data (10 experiments 2009 - 2010

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 0.5% = 1.44 0.6% = 1.87 2.5% = 4.27 EC3 = 1.5%

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: control = 412.0 0.5% = 592.8* 0.6% = 769.6* 2.5% = 1757.8* *: statistically significant increase vs. control group (p<0.05)

Any other information on results incl. tables

Lymph node weight and cell counts were statistically increased in the highest dose (2.5%)

Index LN weights                  LN Cell Count Index

0.5%       1.1                                      1.1

0.6%       1.1                                      1.0

2.5%       1.5                                      1.63

Ear weights were significantly increased in the highest dose (2.5%) - An influence of the irritant property of the test substance on the result of the study cannot be excluded

Ear weight              Index compared to control

0.5%       26.4                            1.0

1.0%       27.2                            1.1

2.5%       30.7*                          1.2

calculated EC3 value:

EC3 = (a-c) [(3-d)/(b-d)] + c = 1.7 % (w/w)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

No symptoms of systemic or local toxicity were observed.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test item was a skin sensitiser under the test conditions of this study.

Executive summary:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone:olive oil (4+1 v/v). For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 0.6% and 2.5% (w/w). The highest concentration tested was the highest concentration that could be technically used and applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by three pre-experiments).

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. However, a statistically significant increase in ear weight was observed in the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice (see Ref. 9) was exceeded in this group (index of 1.2).

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.44, 1.87, and 4.27 were determined with the test item at concentrations of 0.5%, 0.6% and 2.5% (w/w) in acetone:olive oil (4+1 v/v), respectively. A dose response was observed. The EC3 value calculated was 1.5 % (w/w).

A statistically significant increase in DPM value was observed in all test item treated groups in comparison to the vehicle control group (p<0.05), in the mid and high dose group, this increase was also considered as biologically relevant. Furthermore, a statistically significant and biologically relevant increase in lymph node weight and cell count was determined in the highest dose group and the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in this group (index of 1.63). Based on the above mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded. Nevertheless, on the basis of the present data, the test item has to be classified as a sensitiser.