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Endpoint summary

Description of key information

Skin Sensitisaion, Kochan (2018) LLNA

Under the conditions of this study the test material is sensitising to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2018 to 14 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 – 12 weeks
- Weight at study initiation: 17.76 – 20.85 g
- Housing: IVC polycarbonate cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 50 - 60 %
- Photoperiod: 12-hour light / 12-hour dark cycle

Vehicle:

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

Concentration:

100, 50, 25, 2.5 and 1.0 % w/v

No. of animals per dose:

5 females per dose

Details on study design:

TEST MATERIAL PREPARATION AND ADMINISTRATION
- Compound solubility: The vehicle (Acetone/Olive Oil; 4:1; v/v) was selected from the recommended vehicles according to OECD Guideline No. 429. The test material was not soluble in the vehicle; therefore, a homogeneous suspension was obtained.
- Dose preparation: The required amount of the test material (according to the concentration) was mixed with the vehicle shortly before the administration. The test material in the amount of 1000 mg was mixed with the vehicle up to a volume of 1 mL (1000 mg/mL). This was the highest achievable concentration (100 % w/v). The lower concentrations were prepared by mixing of 500, 250, 25 and 10 mg with vehicle up to 1 mL – giving 50, 25, 2.5 and 1.0 % w/v concentration. The preparations were made freshly before each dosing occasion.
25 µL of the test item was applied to the dorsum of each ear. The α-hexyl cinnamaldehyde (25 %) as positive control and vehicle as a negative control were administrated in the same volume.


PRE-SCREEN TESTS:
- All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 %.
- Erythema Scoring:
No erythema = 0
Very slight erythema (barely perceptible) = 1
Well-defined erythema = 2
Moderate to severe erythema = 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema = 4

MAIN STUDY
- Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test material, or the vehicle alone was applied.
- Days 2 and 3: The application procedure carried out on day 1 was repeated.
- Days 4 and 5: No treatment.
- Day 6: The body weight of each animal was recorded. 250 µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 10^4 Bq) of 125I-iododeoxyuridine and 10^-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein. Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).

- Preparation of cell suspensions: Cell suspension of lymph node cells from pooled treatment groups was prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged by 600 g at 4 °C for 10 min. Suspension of cells were precipitated with 5 % trichloroacetic acid (TCA) at 4 °C for 18-20 h. Pellets were centrifuged by 2000 g at 4 °C for 5 min., re-suspended in 1 mL TCA and transferred to gamma counting tubes for 125I-counting.

- Determination of cellular proliferation (incorporated radioactivity): Incorporation of 125I-iododeoxyuridine was measured by 125I-counting on Automatic Gamma Counter Wizard (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/animal. DPM were calculated according to formula:
DPM = CPM / absolute detector efficiency
(Absolute detector efficiency for Gamma Counter Wizard 2470 = 66.73 %)

- Clinical Observation: Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.

- Body Weight: The animal body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals. The significance in difference between initial and terminal body weights was analysed by Student´s T-test; p – value of less than 0.05 were considered significant.

CALCULATION OF RESULTS
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI. A substance is regarded as sensitiser in the LLNA test when SI ≥3.
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation:

EC3=c+[(3-d)/(b-d)]x(a-c)

a – higher concentration, b – SI of higher concentration, c – lower concentration, d – SI of lower concentration

If all points are below the stimulation index of three, no EC3 value can be stated.
When there are no data points that fall below SI = 3, log-linear extrapolation may be applied in which the two lowest test concentrations from the dose-response curve are used, provided the lowest SI value approaches the value of 3 and that a linear dose-response exists. The equation for EC3 calculation:

EC3=2^{log2(c)+ (3−d)/(b−d)×[log2(a)−log2(c)]}

RELIABILITY CHECK
- The reliability of experimental technique used was performed with five females, using α-Hexylcinnamaldehyde (positive control) as a part of the study.
- The same procedure as on treated and control animals were used. A pilot study on the positive control was not necessary due to the fact that the used substance (α-Hexylcinnamaldehyde) is well known.

Positive control substance(s):

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

Positive control results:

The positive control data are included in Table 1.

Key result

Parameter:

EC3

Value:

0.85

Key result

Parameter:

SI

Value:

4.3

Test group / Remarks:

1.0 %

Key result

Parameter:

SI

Value:

11.6

Test group / Remarks:

2.5 %

Key result

Parameter:

SI

Value:

18.6

Test group / Remarks:

25 %

Key result

Parameter:

SI

Value:

25.4

Test group / Remarks:

50 %

Key result

Parameter:

SI

Value:

19.5

Test group / Remarks:

100 %

Cellular proliferation data / Observations:

PRE-SCREEN TEST
- The test material was administered in two mice at a concentration of 100 %. After the test material application, no signs of local irritation at the application site or systemic toxicity were observed.
- The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the test.
- The erythema score (0) and the increase in ear thickness (day 1: 0.193 mm, day 3: 0.198 mm and day 6: 0.193 mm) did not meet the criteria which are considered as signs for excessive local skin irritation (erythema score ≥ 3; ear thickness increase ≥25 %).
For calculation of mean and S.D. values of odyweights and ear thickness MS Excel was used.

MAIN STUDY
- Dose Selection: Based on the observations recorded in the preliminary test, the concentration of 100 % was selected as top dose, and other two concentrations – 50 and 25 % were tested. Because of high SI values at concentrations of 100, 50 and 25 % other two concentrations (2,5 and 1.0 %) were added.
- Clinical Observations: Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show any visible clinical signs.
- Body Weights : The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. The increase of body weight was observed at all used concentrations. The increase was statistically significant at concentrations of 100 and 25 %.
- Lymph Node Proliferation: In comparison with the control group, an increase of the pooled lymph node weights at all concentrations was observed, the increase was dose dependent in the concentration range of 1 – 50 %. The pooled lymph node weights of treated groups were 0.0419 g for 1.0 % concentration, 0.0707 g for 2.5 % concentration, 0.0872 g for 25 % concentration, 0.1046 g for 50 % concentration and 0.0976 g for 100 % concentration of tested material. The lymph node weight of control group and positive control group were 0.0271 and 0.0677 g, respectively. The DPM values for the five treated groups were 2624 (1.0 %), 7069 (2.5 %), 11360 (25 %), 15505 (50 %) and 11933 (100 %), respectively. The SI values for the five treated groups were 4.3 (1.0 %), 11.6 (2.5 %), 18.6 (25 %), 25.4 (50 %) and 19.5 (100 %), respectively. The Lymph node weights, DPM, SI and EC3 value are shown in Table 1.

- Reliability Check : The positive control data are also included in Table 1.

DISCUSSION
- Despite testing five concentrations, there were no data points that fell below SI = 3, therefore for the EC3 value calculation, a log-linear extrapolation was applied.
- As the SI for some treatment dose group is ≥3 and a clear dose response relationship is observed, the test material is regarded as a potential skin sensitiser.

Table 1: The Lymph Node Weight, DPM, SI and EC3 Values.

Treatment (% Concentration)	Lymph Node Weight (g)	Number of Lymph Nodes	DPM	SI	EC3 (%)
Control	0.0271	10	612	-	0.85
Positive Control	0.0677	10	3763	6.15
1.0	0.0419	10	2624	4.3
2.5	0.0707	10	7069	11.6
25	0.0872	10	11360	18.6
50	0.1046	10	15505	25.4
100	0.0976	10	11933	19.5

Interpretation of results:

other: EU Criteria: Category 1A (H317: May cause an allergic skin reaction)

Conclusions:

Under the conditions of this study the test material is sensitising to skin.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 429, under GLP conditions in the Local Lymph Node Assay (LLNA).

Based on the recommendations of the OECD Guideline 429, the test material was suspended in Acetone:Olive Oil 4:1. The positive control (α-Hexylcinnamic aldehyde) (25 %) was dissolved in the same vehicle.

The Pre-screen test was performed using a dose of 100 %. Based on the observations recorded in the Pre-screen tests, the concentration of 100 % was selected as top dose for the main test. 

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test material at concentrations of 100, 50, 25, 2.5 and 1.0 %, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10^-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test material at five concentrations (100, 50, 25, 2.5 and 1.0 % w/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity. In this study the mean Stimulation Indices (SI) of 19.5, 25.4, 18.6, 11.6 and 4.3 were determined with the test material at concentrations of 100, 50, 25, 2.5 and 1.0 % in Acetone:Olive Oil 4:1, respectively. The calculated concentration eliciting SI of 3 (EC3) was 0.85 %.

Under the conditions of this study the test material is sensitising to skin.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In Vitro Skin Sensitisation

In non-GLP pre-tests the solubility of the test material in test specific solvents was determined. The test material was insoluble in all solvents indicated by the respective OECD guidelines at the required concentrations.  Under these circumstances, in vitro skin sensitisation testing in accordance with OECD 442C, OECD 442D, and OECD 442E of the test material is not possible. Testing with precipitates is technically not feasible in the case of DPRA and h-CLAT, and it is not recommended in case of the LuSens test, since a significant result cannot be obtained. Skin sensitisation was therefore assessed in vivo.

In Vivo Skin Sensitisation

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 429, under GLP conditions in the Local Lymph Node Assay (LLNA). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on the recommendations of the OECD Guideline 429, the test material was suspended in Acetone:Olive Oil 4:1. The positive control (α-Hexylcinnamic aldehyde) (25 %) was dissolved in the same vehicle.

The Pre-screen test was performed using a dose of 100 %. Based on the observations recorded in the Pre-screen tests, the concentration of 100 % was selected as top dose for the main test. 

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test material at concentrations of 100, 50, 25, 2.5 and 1.0 %, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10^-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test material at five concentrations (100, 50, 25, 2.5 and 1.0 % w/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity. In this study the mean Stimulation Indices (SI) of 19.5, 25.4, 18.6, 11.6 and 4.3 were determined with the test material at concentrations of 100, 50, 25, 2.5 and 1.0 % in Acetone:Olive Oil 4:1, respectively. The calculated concentration eliciting SI of 3 (EC3) was 0.85 %.

Under the conditions of this study the test material is sensitising to skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified as Category 1A (H317: May cause an allergic skin reaction).