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Description of key information

Skin sensitisation: sensitising, EC3 = 17.9 %, female mice, OECD TG 429, 2019

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09-01-2019 to 29-01-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2015; signature: September 2015
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Microbiological status of animals, when known: Not applicable.
- Age at study initiation: 9-11 weeks
- Weight at study initiation: Definitive Test: 16.4 to 20.2 grams (all females gained body weight during the study).
- Housing: Group housed in Makrolon type II cages (pre-test) and Makrolon III cages (main study) with wire mesh top and granulated soft wood bedding
- Diet (e.g. ad libitum): Certified rodent diet, ad libitum
- Water: mains tap water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: None indicated.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hours light / 12 hours dark

- IN-LIFE DATES: 09-01-2019 to 29-01-2019
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Preliminary test: undiluted (100%), 50%v/v and 25%v/v and 10%v/v in Acetone/Olive Oil (4:1)
- Main test: 25%v/v, 10%v/v and 5%v/v in Acetone/Olive Oil (4:1)
No. of animals per dose:
Preliminary test: 1
Main test: 5 per dose group
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse per test item concentration. The mouse was treated by daily application of 25 µL to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) of the undiluted test item (100% v/v), 50%v/v in acetone olive oil (4:1) and based on the results a further pre-test at 25%v/v and 10%v/v in acetone olive oil (4:1). The mice were at least once daily. Local skin irritation was scored daily according to the scale included in the full study report. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a micrometer, pre dose and 1-hour post dose on Day 1, Days 2 and 3. Additional measurements were taken on Days 4 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser". Additionally, the data must be 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:
Groups of five mice were treated with the undiluted test item or the test item at concentrations in acetone/olive oil 4:1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 19.5 μCi/ml, specific activity 78.1 µCi/mL).

Observations:
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and prior to 3HTdR treatment and Day 6 (prior to termination).
- Ear thickness measurements: The thickness of each ear was measured using a micrometer, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Preparation of Single Cell Suspension:
Draining lymph nodes were rapidly excised and pooled per female (2 nodes per female). 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 approximately 18 hours for precipitation of macromolecules.

Determination of 3HTdR Incorporation:
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships.
Positive control results:
In a concurrent 'positive control study' performed according to OECD TG 429, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde (85%) at 25% v/v in acetone/olive oil 4:1. The highest concentration tested showed a Stimulation Index (SI) of 7.6 and met the criteria for a 'positive' result.

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.

Within the program, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier (DPM value determined for number 7) was detected in the Grubb’s, but not in the Dean-Dixon-Test, and was therefore not excluded from calculations.
Parameter:
SI
Value:
1.5
Test group / Remarks:
5% v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
1.9
Test group / Remarks:
10% v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
4
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
See tables.

DETAILS ON STIMULATION INDEX CALCULATION
The EC3 was interpolated using the expression: EC3 = (a-c) [(3-d)/(b-d)] + c

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)] + c = 17.9%
Where: a = 10% b = 1.9 and c = 25% and d = 4.0

CLINICAL OBSERVATIONS:
There was no signs of clinical/systemic toxicity reported during the study.

BODY WEIGHTS
All females gained bodyweight during the study from measurements taken pre-dose (day 1) to post-termination (day 6).

In the preliminary screening test: On day 1 at 50% and 100% the females showed reduced spontaneous activity. From day 1 to 6 the females showed an erythema of ear skin (score 1 to 3) as well as scaly ears and slight eschar formation on day 6. Redness of the scalp was observed on day 1 and 2 at 100%v/v test item concentration. At 10% and 25% the tested concentrations did not show any signs of systemic toxicity and from day 1 to 4 the females showed an erythema of the skin (score = 1). At 25%v/v the female showed scaly ears on day 6. Based on the preliminary test concentrations of 25%, 10% and 5%v/v were selected for the main test. All preliminary test females gained (or maintained) body weight during the period.

 

In the definitive test, there were no deaths or signs of systemic toxicity. Body weights were comparable to that observed in the corresponding control group over the same period. The disintegrations per minute (DPM) and stimulation index (SI) are given in the table for all test item concentrations. During the definitive test erythema were observed (score = 1) during the study days 2 to 5.

 

Table 1.0 – Individual Disintegrations per Minute and Stimulation Index during the test

Test item concentration

DPM values measured

DPM-BG per animal
(2 lymph nodes) a)

S.I. b)

Group Mean DPM per individual (2 lymph nodes)

S.D.

Group Mean S.I.

%

Group no.

Individual no.

---

---

BG I

34

---

---

 

 

 

---

---

BG II

22

---

---

 

 

 

Control 0

1

1

581

553

---

 

 

 

Control 0

1

2

635

607

---

 

 

 

Control 0

1

3

1214

1186

---

 

 

 

Control 0

1

4

1138

1110

---

 

 

 

Control 0

1

5

1486

1458

---

982.8

390.3

1.0

5

2

6

1583

1555

1.6

 

 

 

5

2

7

1255

1227

1.2

 

 

 

5

2

8

1594

1566

1.6

 

 

 

5

2

9

1493

1465

1.5

 

 

 

5

2

10

1628

1600

1.6

1482.6

151.3

1.5

10

3

11

1085

1057

1.1

 

 

 

10

3

12

2280

2252

2.3

 

 

 

10

3

13

2404

2376

2.4

 

 

 

10

3

14

2026

1998

2.0

 

 

 

10

3

15

1656

1628

1.7

1862.2

533.3

1.9

25

4

16

2861

2833

2.9

 

 

 

25

4

17

4348

4320

4.4

 

 

 

25

4

18

4349

4321

4.4

 

 

 

25

4

19

4815

4787

4.9

 

 

 

25

4

20

3215

3187

3.2

3889.6

834.7

4.0 #

HCA 25

5

21

7097

7069

7.2

 

 

 

HCA 25

5

22

8609

8581

8.7

 

 

 

HCA 25

5

23

6275

6247

6.4

 

 

 

HCA 25

5

24

8832

8804

9.0

 

 

 

HCA 25

5

25

6919

6891

7.0

7581.4

1117.4

7.6 #

 

 

 

 

 

 

 

 

 

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

BG=Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group for the test item and for the positive control item

2-4 = Test Groups           

5 = Positive Control Group

S.I. =Stimulation Index

a) = values corrected for mean background value (BGI and BGII)

b) = Stimulation Indices relative to the mean of the control group (Group 1)

# = statistically significant versus control

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the condition of this study, the test item is considered to be sensitising to skin. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 17.9%.
Executive summary:

The study was performed to OECD TG 429 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test single mice were treated by daily application of 25 μl of the test item at specified concentrations to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. The preliminary test was conducted: undiluted (100%v/v) and at 50%v/v in acetone/olive oil (4:1) vehicle. A follow up preliminary test at 25%v/v and 10%v/v was also conducted. At 50%v/v and 100%v/v the females showed reduced spontaneous activity. From day 1 to 6 the females showed an erythema of ear skin (score 1 to 3) as well as scaly ears and slight eschar formation on day 6. Redness of the scalp was observed on day 1 and 2 at 100%v/v test item concentration. At 10%v/v and 25%v/v the tested concentrations did not show any signs of systemic toxicity and from day 1 to 4 the females showed an erythema of the skin (score = 1). At 25%v/v the female showed scaly ears on day 6. All preliminary test females gained (or maintained) body weight during the period. Based on the preliminary test concentrations of 25%v/v, 10%v/v and 5%v/v were selected for the main test. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered and spread over the dorsal surface of the ear. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual animal of each group the draining auricular lymph nodes were excised and processed. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensitising if at least one concentration of the substance resulted in a 3-fold or greater increase in 3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights were comparable to controls. A stimulation index (SI) of more than 3 was noted at the 25%v/v test concentration. The 25%v/v group generated a SI = 4.0. The EC3 value was calculated by linear interpolation to be 17.9%. The positive control gave a stimulation index > 3.0 thereby demonstrating the sensitivity and reliability of the test system. Accordingly, the test item was considered to be sensitising under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin Sensitisation:

Key study : in vivo: OECD TG 429, 2019 : The study was performed to OECD TG 429 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test single mice were treated by daily application of 25 μl of the test item at specified concentrations to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. The preliminary test was conducted: undiluted (100%v/v) and at 50%v/v in acetone/olive oil (4:1) vehicle. A follow up preliminary test at 25%v/v and 10%v/v was also conducted. At 50%v/v and 100%v/v the females showed reduced spontaneous activity. From day 1 to 6 the females showed an erythema of ear skin (score 1 to 3) as well as scaly ears and slight eschar formation on day 6. Redness of the scalp was observed on day 1 and 2 at 100%v/v test item concentration. At 10%v/v and 25%v/v the tested concentrations did not show any signs of systemic toxicity and from day 1 to 4 the females showed an erythema of the skin (score = 1). At 25%v/v the female showed scaly ears on day 6. All preliminary test females gained (or maintained) body weight during the period. Based on the preliminary test concentrations of 25%v/v, 10%v/v and 5%v/v were selected for the main test. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered and spread over the dorsal surface of the ear. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual animal of each group the draining auricular lymph nodes were excised and processed. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensitising if at least one concentration of the substance resulted in a 3-fold or greater increase in 3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights were comparable to controls. A stimulation index (SI) of more than 3 was noted at the 25%v/v test concentration. The 25%v/v group generated a SI = 4.0. The EC3 value was calculated by linear interpolation to be 17.9%. The positive control gave a stimulation index > 3.0 thereby demonstrating the sensitivity and reliability of the test system. Accordingly, the test item was considered to be sensitising under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation category 1B: H317

 

The weight of evidence indicates that the substance has a low frequency of occurrence in humans and/or low to moderate potency in animals (EC3 >2%) and can be presumed to have the potential to produce sensitisation in humans via the dermal route.