Registration Dossier

Administrative data

Description of key information

Skin sensitisation: sensitising, EC3 = 22.0 %, female mice, OECD TG 429, 2015

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:
14-10-2014 to 24-03-2015
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2014; signature: May 2014
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised Supplier
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (ad libitum): 2014C Teklad Global Rodent diet (Recognised Supplier); ad libitum
- Water (ad libitum): mains tap water; ad libitum
- Acclimation period: at least 5 days

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

IN-LIFE DATES: Fro: 06-02-2012 To: 28-02-2012
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Preliminary test: 100% w/w
- Main test:
EXP 1: 100%, 50%, 25% w/w ; An additional test was conducted as all concentrations yielded positive in EXP1
EXP 2: 25%, 10% and 1% w/w
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 substance, a preliminary screening test was performed using one mouse per test item concentration. The mouse was treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 100% w/w undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. 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 gauge, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 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. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema was noted on both ears on Days 2 and 3 and a 6.7% body weight loss was also noted. On this basis 100%, 50%, 25% w/w concentrations was selected for the main test.

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".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the test item at concentrations of: EXP 1: 100%, 50%, 25% w/w (an additional test was conducted as all concentrations yielded positive in EXP1) and EXP 2: 25%, 10% and 1% w/w in acetone/olive oil 4:1. A further group of five mice received the vehicle alone in the same manner. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. 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 using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

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:80μCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 μCi to each mouse.

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 Day 6 (prior to termination).
- Ear thickness measurements: The thickness of each ear was measured using a gauge, pre-dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Daily mean ear thickness changes were calculated. 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. Due to technician error, the Day 1 post-dose ear thickness measurements were not performed. This deviation from the study plan was considered not to affect the purpose or integrity of the study.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately 18 hours incubation at approximately 4 degrees Celsius, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using a recognised scintillation system.
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. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
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 7.19 and met the criteria for a 'positive' result.
The results of routine positive control testing (Historic Control Data) performed according to OECD TG 429, using α-hexylcinnamaldehyde (85%) and Phenylacetaldehyde (90%) indicated positive results using a variety of different vehicles. Information is documented in the full study report.
Parameter:
SI
Value:
3.36
Test group / Remarks:
25% w/w in acetone/olive oil 4:1
Parameter:
other: disintegrations per minute (DPM)
Value:
6 561.92
Variability:
±2717.95
Test group / Remarks:
25% w/w in acetone/olive oil 4:1
Remarks on result:
other: See table.
Parameter:
SI
Value:
1.54
Test group / Remarks:
10% w/w in acetone/olive oil 4:1
Parameter:
SI
Value:
1.55
Test group / Remarks:
1% w/w in acetone/olive oil 4:1
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The EC3 was determined based on the equation: EC3 = c + [[(3−d)/(b−d)] x (a−c)]

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as negative (vehicle) controls over the study period. A greater than expected body weight loss (3.7 g) was noted in one positive control animal.

Table 1: Experiment 2: Stimulation Index (mean radioactive incorporation for each group divided by mean radioactive incorporation of vehicle control group).

Concentration (% w/w) in acetone/olive oil 4:1

Stimulation Index

Result

1

1.55

Negative

10

1.54

Negative

25

3.36

Positive

Table 2: Experiment 2: Individual Disintegrations per Minute and Stimulation Indices 

 

Treatment Group

Individual Number

dpm/ Individual #a

Mean dpm/individual

(Standard Deviation)

Stimulation

Index

#b

 

Result

Vehicle acetone/olive oil

4:1

1-1

1281.19

1950.77 (±851.53)

na

na

1-2

3085.53

1-3

1230.85

1-4

2632.06

1-5

1524.22

Test Item

1% v/v in acetone/olive oil

4:1

2-1

2459.42

3025.68 (±907.54)

1.55

Negative

2-2

4135.48

2-3

3791.00

2-4

1984.02

2-5

2758.46

Test Item

10% v/v in acetone/olive oil

4:1

3-1

3207.33

3012.33 (±1705.57)

1.54

Negative

3-2

1543.90

3-3

5770.13

3-4

1664.18

3-5

2876.13

Test Item

25% v/v in acetone/olive oil

4:1

4-1

6126.70

6561.92** (±2717.95)

3.36

Positive

4-2

6865.03

4-3

5790.08

4-4

3254.67

4-5

10773.11

Positive Control

Item

25% v/v in acetone/olive oil

4:1

5-1

13364.42

14024.29** (±2103.42)

7.19

Positive

5-2

12160.83

5-3

17410.30

5-4

12604.92

5-5

14580.99

dpm = Disintegrations per minute

a = Total number of lymph nodes per individual is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

** = Significantly different from vehicle control group p < 0.01

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 substance expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 22%.
Executive summary:

The study was performed to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600, Local Lymph Node Assay 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 solid test item diluted at 100% 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. Based on the preliminary test, the concentrations selected for the main test were 0% (control), 25%, 50% within Acetone/Olive Oil (4:1) and 100% w/w. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration (100% w/w).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 using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated daily 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. For each individual animal 1 mL of PBS was added to the lymph nodes. Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured. 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 in3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. Due to positive results at all concentrations in the initial test, a further test was completed at 0% (control), 1%, 10% and 25% within Acetone/Olive Oil (4:1). In the main test, there were no deaths or signs of systemic toxicity, and body weights in treatment groups were comparable to controls. A stimulation index (SI) was recorded for each concentration as follows: 1%w/w: SI = 1.55, 10%w/w: SI = 1.54 and 25%w/w: SI = 3.36. The EC3 was calculated to be 22%. 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, 2015 : The study was performed to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600, Local Lymph Node Assay 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 solid test item diluted at 100% 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. Based on the preliminary test, the concentrations selected for the main test were 0% (control), 25%, 50% within Acetone/Olive Oil (4:1) and 100% w/w. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration (100% w/w).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 using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated daily 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. For each individual animal 1 mL of PBS was added to the lymph nodes. Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured. 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 in3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. Due to positive results at all concentrations in the initial test, a further test was completed at 0% (control), 1%, 10% and 25% within Acetone/Olive Oil (4:1). In the main test, there were no deaths or signs of systemic toxicity, and body weights in treatment groups were comparable to controls. A stimulation index (SI) was recorded for each concentration as follows: 1%w/w: SI = 1.55, 10%w/w: SI = 1.54 and 25%w/w: SI = 3.36. The EC3 was calculated to be 22%. 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.