Phosphorous ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-04-09 to 2009-04-15

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

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:

esterification products of castor oil and polyphosphoric acids

EC Number:

614-406-6

Cas Number:

68308-61-2

Molecular formula:

C57H107P3O18

IUPAC Name:

esterification products of castor oil and polyphosphoric acids

Details on test material:

- Name of test material (as cited in study report): GARDO TP10451
- Physical state: liquid
- Analytical purity: 100%
- Lot/batch No.: 2939R
- Expiration date of the lot/batch: 2010-02-18

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: WOBE Kereskedelmi Kft.H-1164, Budapest, Garmada u. 10.
- Age at study initiation: Young adult, 9-10 weeks old, age-matched within one week
- Weight at study initiation: The weight variation in animals involved in the study did not exceed  20 % of the mean weight.
- Cage type: Type II. polypropylene/ polycarbonate
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 8-12 air exchange/hour by central air-condition system.
- Photoperiod (hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

50%, 25% and 10%

No. of animals per dose:

4 animals/treatment group

Details on study design:

In the main assay sixteen female CBA/Ca mice were allocated to four groups of four animals each:
- three groups received the appropriate formulation of GARDO TP10451 at concentrations of 50 %, 25 % or 10 %,
- the negative control group received the solvent Acetone: Olive oil 4:1 mixture (AOO).
Each substance was applied on the external surface of each ear (25 µL/ear) for three consecutive days (Day 0, 1 and 2) at the appropriate concentrations. On Day 5, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl-thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

Positive control substance(s):

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

Statistics:

NA

Results and discussion

Positive control results:

The positive control substance alpha-Hexylcinnamaldehyde (HCA) was examined at a concentration of 25 % in the relevant vehicle. A significant lymphoproliferative response (SI >= 3) was noted for HCA with stimulation index value of 11.3 in accordance with our previous data, demonstrating the test reliability.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

A lymphoproliferative response (SI >= 3) was noted for GARDO TP10451 at concentrations of 50 % and 25 %. No lymphoproliferative response was observed at concentration of 10 %. The stimulation index values were 3.1, 3.1 and 1.3 at concentrations of 50 %, 25 % and 10 %, respectively. The stimulation index values were compatible with the conventional biological dose-response.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: NA

Any other information on results incl. tables

Clinical Signs and Mortality:

No mortality or signs of systemic toxicity were observed during the study. No treatment related effects were observed on animal body weights. Slight irritation (redness at the base of the ears) with recovery was observed in all of the test item treated groups with dose dependent intensity. No other cutaneous effect was observed.

Proliferation Assay:

A lymphoproliferative response (SI >= 3) was noted for GARDO TP10451 at concentrations of 50 % and 25 %. No lymphoproliferative response was observed at concentration of 10 %. The stimulation index values were 3.1, 3.1 and 1.3 at concentrations of 50 %, 25 % and 10 %, respectively. The stimulation index values were compatible with the conventional biological dose-response.

Reliability of the Assay:

The result of the latest reliability check was used to demonstrate the appropriate performance of the assay in accordance with the OECD guideline. The positive control substance alpha-Hexylcinnamaldehyde (HCA) was examined at a concentration of 25 % in the relevant vehicle. A significant lymphoproliferative response (SI >= 3) was noted for HCA with stimulation index value of 11.3 in accordance with our previous data, demonstrating the test reliability.

Interpretation of Observations:

The maximum concentration of test item in an acceptable solvent was established according to the principles of the relevant guidelines. The maximum available concentration based on solubility was 50 % (% w/v) in Acetone : Olive oil 4:1 mixture (AOO) and in N,N-Dimethylformamide (DMF). Since AOO is the preferred solvent by the relevant guideline, this solvent was selected to be used. Although loss of hair was observed with test item concentration of 50 % in the preliminary test it was not significant, so to meet the requirement of the OECD 429 guideline to test as high concentration as possible 50 % was accepted as the maximum.

A lymphoproliferative response (SI >= 3) was noted for GARDO TP10451 at concentrations of 50 % and 25 %. No lymphoproliferative response was observed at concentration of 10 %. The proliferation values obtained corresponded to the conventional biological dose-response.

Although irritation was observed in the main assay it was not excessive. It is known that some non-sensitising irritant chemicals (such as sodium dodecyl sulphate) can give a slight positive response in the LLNA but there are also many irritants which do not give such a response. It is plausible that the slight increase in proliferation seen in this study could be secondary to the irritation effect, but there is no specific evidence to make this argument. So in line with the guidelines, the test item must be considered to be positive.

The EC3 value (the theoretical concentration of the test item in the test solution, leading a three fold increase of lymph node cell proliferation over the control) was estimated by linear interpolation using the reported SI values and the corresponding concentrations immediately above and below the SI value of 3. The estimated EC3 value of GARDO TP10451 was 24.2 % in this LLNA. On the basis of published classification of contact allergens according to their potency GARDO TP10451 can be ranked among weak sensitisers (EC3 (%)

Applicant's summary and conclusion

Interpretation of results:

other: weak-sensitiser

Conclusions:

Under the conditions of the present assay GARDO TP10451, dissolved in an appropriate solvent up to the maximum feasible concentration (50 %), was shown to have weak sensitisation potential (weak-sensitiser) at slightly irritating concentrations in the Local Lymph Node Assay.
Thus, the substance is to be classified as "Sensitizing by skin contact" (R43). The registrant proposes to perform an in vivo study to verify this classification.

Executive summary:

A lymphoproliferative response (SI >= 3) was noted for GARDO TP10451 at concentrations of 50 % and 25 %. No lymphoproliferative response was observed at concentration of 10 %. The stimulation index values were 3.1, 3.1 and 1.3 at concentrations of 50 %, 25 % and 10 %, respectively. The stimulation index values were compatible with the conventional biological dose-response. No mortality or systemic clinical signs were observed during the study. No treatment related effect on the body weights was observed.

Slight irritation (redness at the base of the ears) with recovery was observed in all of the test item treated groups with dose dependent intensity. No other cutaneous effect was observed.

Thus, under the conditions of the present assay GARDO TP10451, dissolved in an appropriate solvent up to the maximum feasible concentration (50 %), was shown to have weak sensitisation potential (weak-sensitiser) at slightly irritating concentrations in the Local Lymph Node Assay.