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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:
14-10-2014 to 28-10-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met or with minor deviations considered acceptable for assessment.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: March 2014 ; signature: May 2014
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Reaction mass of (4E,8Z)-cyclododeca-4,8-dien-1-one and (4Z,8E)-cyclododeca-4,8-dien-1-one and (4E,8E)-cyclododeca-4,8-dien-1-one
EC Number:
941-893-5
Cas Number:
15229-79-5
Molecular formula:
C12H18O
IUPAC Name:
Reaction mass of (4E,8Z)-cyclododeca-4,8-dien-1-one and (4Z,8E)-cyclododeca-4,8-dien-1-one and (4E,8E)-cyclododeca-4,8-dien-1-one
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark under nitrogen
- Other: clear colourless

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: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Microbiological status of animals, when known: Not applicable.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15 - 23 grams ; Preliminary Test : 18.9 g ; Definitive Test: 15.3 to 20.0 grams (all females treated with test item gained body weight during the study ; where losses occurred they were within typical range for the species and strain, without dose response).
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- 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: 14-10-2014 to 28-10-2014

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Preliminary test: undiluted (100%)
- Main test: undiluted (100%), 50%v/v and 25%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 of the undiluted test item or the test item at a concentration of 100% v/v, 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 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". Applicant assessment also indicates: 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 of 50% or 25% v/v in acetone/olive oil 4:1.The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest selected 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 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:
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. Data was also assessed for outliers using the Grubb's test.

Results and discussion

Positive control results:
In a non-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 8.38 and met the criteria for a 'positive' result. The non-concurrent 'positive control study' dates were reported in the full study report (conducted within 6 months of the definitive test).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks:
mean (n=4)
Value:
3.11
Test group / Remarks:
25% v/v in acetone:olive oil (4:1)
Remarks on result:
other: Result based on four females (n=4) due to exclusion of outlier value
Remarks:
2-5] removed as statistically significant outlie via Grubb's test
Parameter:
SI
Remarks:
mean (n=5)
Value:
3.95
Test group / Remarks:
50% v/v in acetone:olive oil (4:1)
Parameter:
SI
Remarks:
mean (n=5)
Value:
6.69
Test group / Remarks:
100% test item
Parameter:
other: disintegrations per minute (DPM)
Remarks:
mean (n=4)
Value:
2 434.05
Variability:
±227.00
Test group / Remarks:
25% v/v in acetone:olive oil (4:1)
Remarks on result:
other: See table below
Remarks:
2-5] removed as statistically significant outlie via Grubb's test
Parameter:
other: disintegrations per minute (DPM)
Remarks:
mean (n=5)
Value:
3 096.92
Variability:
±1036.94
Test group / Remarks:
50% v/v in acetone:olive oil (4:1)
Parameter:
other: disintegrations per minute (DPM)
Remarks:
mean (n=5)
Value:
5 238.8
Variability:
±859.71
Test group / Remarks:
100% test item
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
See tables. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per individual and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

EC3 CALCULATION
The EC3 was by applicant calculation: extrapolated using the expression :
EC3 = 2 ^{log2(c)+(3-d)/(b-d)x[log2(a)-log2(c)]}
Where:
a = mid concentration giving a stimulation index > 3 ; = 50%
b = actual stimulation index caused by ‘a’ ; = 3.95
c = lowest concentration giving a stimulation index of > 3 ; = 25%
d = actual stimulation index caused by ‘c’ ; = 3.11
Reference: Ryan, C. et al., Extrapolating Local Lymph Node Assay EC3 Values to Estimate Relative Sensitizing Potency. Cutaneous and Ocular Toxicology, 26: 135–145. (2007).

Calculation:
EC3 = 2 ^{log2(c)+(3-d)/(b-d)x[log2(a)-log2(c)]}
EC3 = 2 ^{log2(25)+(3 - 3.11)/(3.95 - 3.11)x[log2(50)-log2(25)]}
EC3 = 2 ^{log2(25) + ( - 0.11) / (0.84) x [log2(50)-log2(25)]}
EC3 = 2 ^{4.64385618977 + ( - 0.11) / (0.84) x [5.64385618977 - 4.64385618977]}
EC3 = 2 ^{4.64385618977 + ( - 0.11) / (0.84) x 1}
EC3 = 2 ^{4.64385618977 + ( - 0.11) / 0.84}
EC3 = 2 ^{4.64385618977 -0.13095}
EC3 = 2 ^{4.5129}
EC3 = 22.83%

CLINICAL OBSERVATIONS:
There were no signs of clinical/systemic toxicity reported during the study. There were no signs of local skin irritation (maximum erythema score = 0). In the preliminary test (100% undiluted) very slight erythema (score = 1) was noted on both ears on Day 2.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period. All females gained bodyweight or had acceptable body weight declines during the study from measurements taken pre-dose (day 1) to post-termination (day 6). Where losses occurred, they were within typical range for the species and strain, without dose response.

Any other information on results incl. tables

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

Test item concentration

DPM values measured

Mean DPM per individual

S.D.

Group Mean S.I.

%

Group no.

Individual no.

(Control) 0

1

1

620.87

 

 

 

(Control) 0

1

2

613.45

 

 

 

(Control) 0

1

3

480.02

 

 

 

(Control) 0

1

4

113.60

 

 

 

(Control) 0

1

5

1068.57

783.50

296.65

n/a

25

2

1

2188.80

 

 

 

25

2

2

2556.12

 

 

 

25

2

3

2305.98

 

 

 

25

2

4

2685.31

 

 

 

25

2

5

5429.90 #1

2343.05

*

 

[3033.22

**]

227.00

 

 

[1354.13]

3.11 #2

 

 

[3.87]

50

3

1

1980.60

 

 

 

50

3

2

4170.43

 

 

 

50

3

3

4020.55

 

 

 

50

3

4

3231.56

 

 

 

50

3

5

2081.47

3096.92 **

1036.94

3.95

100

4

1

4731.18

 

 

 

100

4

2

3969.38

 

 

 

100

4

3

5702.15

 

 

 

100

4

4

5965.43

 

 

 

100

4

5

5825.85

5238.80 ***

859.71

6.69

 

 

 

 

 

 

 

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

a = Total number of lymph nodes per individual is 2

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

n/a = Not applicable

#1 = Considered an outlier, statistically identified via Grubb’s test

#2= Result based on four individuals due to exclusion of outlier value

[ ] = Result including outlier value

* = Significantly different from control group p <0.05

** = Significantly different from control group p <0.01

*** = Significantly different from control group p <0.001

Applicant's summary and conclusion

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 22.8%.
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 was treated by daily application of 25 μl of the test item undiluted (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 or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema was noted on Days 2 to 5. Based on the preliminary test, the concentrations selected for the main test were 0% (control), 25%, 50% and 100% v/v (undiluted). The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration (100% v/v; undiluted). 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 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. 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%, 50% and 100% test concentrations. The 25% v/v group generated a SI = 3.11 (excluding statistically significant outlier in dose group). The 50% v/v group generated an SI = 3.95 and the 100% (undiluted) group an SI = 6.69. The present study was found to have an unusually low mean DPM/node value for the vehicle control group of 391.75, which was lower than the value at the 2.5 percentile compared to the laboratory historical control data (collated dates provided in the full study report). The Mean DPM/lymph node values for the vehicle control group individuals of this study could be considered to be lower than the ‘acceptable’ range. The positive control sensitivity check (conducted within 6 months of the definitive test) gave a stimulation index > 3.0 thereby demonstrating the sensitivity and reliability of the test system. The test item EC3 value was calculated by the applicant by log-linear extrapolation of the data, according to: Ryan, C. et al., Extrapolating Local Lymph Node Assay EC3 Values to Estimate Relative Sensitizing Potency. Cutaneous and Ocular Toxicology, 26: 135–145. (2007). The EC3 value was determined to be 22.8%. Accordingly, the test item was considered to be sensitising under the conditions of the test.

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