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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:
07 November - 27 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with International guidelines and in accordance with GLP. Guideline validity criteria were satisfied.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 29-65 % instead of 45–65%.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 29-65 % instead of 45–65%.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
Although the test item used within this test contains different identifiers as compared to the subtance that is being registered, the tested substance is within the boundary composition of the substance being registered. Therefore, this particular batch of the tested substance, can be considered as the same substance as (Z,Z)-di-9-octadecenyl phosphonate. It can be considered that all tests conducted on Reaction products of triphenyl phosphite, oleyl alcohol and alcohols, C10-16, Batch 2955, are considered relevant and reliable for (Z,Z)-di-9-octadecenyl phosphonate.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: confidential
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable): n/a
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in acetone/olive oil (4+1 v/v) - assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was formulated in 4:1 acetone olive oil (4:1 v/v) prior to use.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: 18.2 – 25.4 g
- Housing: Makrolon Type II (pre-test) / III (main study), with wire mesh top. Granulated soft wood bedding.
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet
 (certified), ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 45 - 65 %
- Air changes (per hr): At least 15 changes/ hour
- Photoperiod (hrs dark / hrs light): 12 h: 12 h (light: dark)
- IN-LIFE DATES: Not reported

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5, and 10%
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: A solubility trial was conducted in cetone/olive oil (4+1 v/v) . The preperations were considered appropriate for the test and this solvent was used in the definitive test.
- Irritation: Yes, unaaceptable levels of irritation were seen in administered concntrations of 100, 50 and 25 %. 10 % was acceptable, as such 10, 5 and 2.5 % was used for the definitive test
- Systemic toxicity: None during the test
- Ear thickness measurements: yes, the dorsum of each ear
- Erythema scores:
/ = No visible erythema
1 = Very slight erythema
2 = Well defined erythema
3 = Moderate to severe erythema
4 = Severe erythema to formation of eschar which prevents grading of erythema

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA OECD 429.
- Criteria used to consider a positive response: EC3

TREATMENT PREPARATION AND ADMINISTRATION:

There were 3 groups of 4 animals in each test group and the control. The test groups were, 2.5, 5, 10 %.
The test item was placed into an appropriate container on a tared balance and acetone/olive oil (4+1 v/v) was added (weight per weight).
The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1 v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20 μCi of 3H-methyl thymidine (equivalent to 80.1 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm 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 scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β- 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:
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Results and discussion

Positive control results:
The animals treated with the positive control item alpha-Hexylcinnamaldehyde (25% (w/v)) showed a distinctly positive response (mean S.I. of 10.33), corroborating the validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 2.29
Test group / Remarks:
Group 2 - 2.5 %
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
ca. 1.18
Test group / Remarks:
Group 3 - 5 %
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
ca. 2.73
Test group / Remarks:
Group 4 - 10 %
Remarks on result:
other: Negative
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: 2.5 % at 10 % concentration.

DETAILS ON STIMULATION INDEX CALCULATION: Mean treatment group dpm / mean vehicle dpm

EC3 CALCULATION: Not applicable, all stimulation indices were < 3 therefore no EC3 was calculable.

CLINICAL OBSERVATIONS: No signs of systemic toxicity or other clinical signs

BODY WEIGHTS: Within normal limits

Any other information on results incl. tables

During the test the temperature was between 19 to 24 °C instead of 20 to 24 °C for several hours; this is only a study plan deviation and within the guideline recommendations of 22 ± 3 °C.

Table 2. Calculation of Stimulation Indices per Dose Group

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM- BGa

number of lymph nodes

DPM perb)lymph node

S.I.

---

BG I

9

---

---

---

---

---

BG I

15

---

---

---

---

0

1

6141

6129

8

766.1

1.00

2.5

2

14059

14047

8

1755.9

2.29

5

3

7273

7261

8

907.6

1.18

10

4

16769

16757

8

2094.6

2.73

1 = Control Group

2-4 = Test Group

a) = The mean value was taken from the figures BGI and BGII

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the condition of the study, the test item, AD-464 did not meet the criteria for skin sensitisation in accordance with the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008.
Executive summary:

OECD 429 (2018): Three groups each of four female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% 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 two pre-experiments. A control group of four mice was treated with the vehicle (acetone/olive oil (4+1 v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β- scintillation counter.

No mortality was reported and no signs of systemic toxicity were observed. Some animal some animals showed an erythema of the ear skin with statistically significant and biologically relevant increase in ear thickness.

Stimulation Indices of 2.29, 1.18, 2.73 were determined with the test item at concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1 v/v). The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

The animals treated with the positive control item alpha-Hexylcinnamaldehyde (25% (w/v)) showed a distinctly positive response (mean S.I. of 10.33), corroborating the validity of the assay.

Based on the condition of the study, the test item did not meet the criteria for skin sensitisation in accordance with the Globally Harmonized Classification System and to CLP Regulation (EC) No. 1272/2008.