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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July- August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was >95% instead of 45 – 65% for several hours. This deviation to the study plan, however, does not affect the validity of the study.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Batch No.: 80627X065A

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Number of animals per group:4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age: 1st pre-test: 10 - 11 weeks (beginning of treatment)
2nd pre-test: 10 - 11 weeks (beginning of treatment)
Main study: 9 – 10 weeks (beginning of treatment)
Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
relative humidity 45-65% (for several hours: >95%)
artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4+1, v/v).
No. of animals per dose:
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone:olive oil (4+1, v/v) as vehicle.
- Irritation: For both concentrations tested 50 and 100%, an increase in ear thickness above the threshold value of 25% was observed on the 6th day of treatment. Compared to historical vehicle data, ear weights were also highly increased in both animals. Moreover, the animal treated with the undiluted test item showed a slight to severe erythema of the ears. The animal treated with a test item concentration of 50% developed an erythema of the ear skin as well. In both animals, a visible swelling of the ears was observed.
Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. For both concentrations, no signs of systemic toxicity or excessive local skin irritation were observed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

TREATMENT PREPARATION AND ADMINISTRATION:
- application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4+1, v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diam. ap. 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
- administration: Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.5 µCi of 3HTdR (equivalent to 3HTdR 81.9 µCi/mL) were injected into each test and control mouse via the tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Experiment performed in April 2012
Test item concentration 10% S.I.: 1.51
Test item concentration 25% S.I.: 3.73

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
control
Remarks on result:
other: see below
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
1.45
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
3.42
Test group / Remarks:
25% (w/v)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see below

Any other information on results incl. tables

table of results:

Test item concentration % (w/v)

Measurement DPM

Calculation

Result

number of lymph nodes

DPM per lymph node

S.I.

 

0

3038

8

377.1

1.00

 

5

3051

8

378.8

1.00

 

10

4398

8

547.1

1.45

 

25

10332

8

1288.9

3.42

 

Calculation of the EC3 value:

 

Test item concentration %

S.I.

Test Group 3

10 (a)

1.45 (b)

Test Group 4

25 (c)

3.42 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c =21.8%(w/v)

other findings:

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item Phosphorous acid, tri-C12-14-alkyl esters was found to be a skin sensitiser under the test conditions of this study.
Executive summary:

Phosphorous acid, tri-C12-14-alkyl esters was tested in a LLNA test for sensitisation. The study followed the current guidelines (OECD 429) and the small deviation did not affect the validity that was quoted to reliability 1 according to Klimlisch criteria.

Three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (acetone:olive oil (4+1, v/v)) only.

The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

In this study Stimulation Indices of 1.00, 1.45 and 3.42 were determined with the test item at concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4+1, v/v). A clear dose response was observed. The EC3 value calculated was 21.8%, which indicates a moderate sensitising potential.