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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 08, 2015 to November 04, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline No. 429 without any deviation
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

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
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on June 17, 2015/ signed on September 24, 2015)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Juniper, Juniperus mexicana, ext., epoxidized
EC Number:
309-066-8
EC Name:
Juniper, Juniperus mexicana, ext., epoxidized
Cas Number:
99811-75-3
Molecular formula:
not applicable (UVCB substance)
IUPAC Name:
(1S,2R,5S,7R)-2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undecan-9-one; (1aS,2R,4aS,8aS)-2,4a,8,8-tetramethyl-decahydrocyclopropa[e]naphthalen-3-one; (1aS,2S,4aS,8aS)-2,4a,8,8-tetramethyl-decahydrocyclopropa[e]naphthalen-3-one
Test material form:
other: liquid
Details on test material:
- Physical state: Pale yellow liquid
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: 15-23g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Standard rodent diet (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: The rate of air exchange was approximately 15 changes/ h.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES:
From October 08, 2015 to November 04, 2015

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 25, 50 % v/v in acetone/olive oil 4:1 and undiluted
No. of animals per dose:
5
Details on study design:
PRELIMINARY STUDY:
- Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- Based on the results of this preliminary test, the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the SI is 3 or more, the test substance is regarded as a skin sensitizer with a consideration given to dose response and statistical significance.

TREATMENT PREPARATION AND ADMINISTRATION:
- For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced a solution at the required 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 to 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. A further group of five mice received the vehicle alone in the same manner. The thickness of each ear was measured and recorded as described in the preliminary screening test. Local skin irritation was scored daily. Five days following the first topical application of the test item (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and placed in 1 mL of PBS. A single cell suspension of the lymph node cells (LNC) for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The LNC were rinsed through the gauze with 9 mL of PBS, transferred to a centrifuge tube, pelleted at 1400 rpm (approximately 190 g) for 10 minutes and resuspended in 10 mL of PBS and re-pelleted. The pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA). After approximately 18 hours incubation at approximately 4 °C, the precipitate was recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA 5% and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The proliferation response of LNC was expressed as the number of radioactive disintegrations per minute per animal (dpm/animal) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

- Stimulation Index: Results for each treatment group were expressed as the Stimulation Index (SI).
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.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

Positive control results:
SI for the positive control substance α-Hexylcinnamaldehyde (HCA) 25% v/v in vehicle, was 5.83 which demonstrates the validity of this study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
- Stimulation index for 25 and 50 % v/v and undiluted material were 3.10, 8.85 and 17.83, respectively. - As a SI of 3 or more was recorded for all of the concentrations tested, test item was considered to have the potential to cause skin sensitization. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3 value) was calculated to be 24.7%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal for 25, 50 % v/v and undiluted material were, 6870.78, 19637.09 and 39532.59, respectively.

Any other information on results incl. tables

Preliminary investigation

Mortality and clinical signs: There were no deaths and no signs of systemic toxicity was noted during the study.

Dermal reactions: Very slight erythema was noted on both ears of the animals receiving undiluted test item on Day 3 (score of 1/4).

Measurement of ear thickness: No signs of irritation indicated by the increase in mean ear thickness were noted.

Body weight : There was no indication of an effect of treatment on body weight gain. A minor loss in body weight was noted

Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

 

Main phase

Mortality and clinical signs: There were no deaths and no signs of systemic toxicity were noted in the test or control animals during the test.

Dermal reactions: No signs of dermal irritation were seen on the ear during the study.

Measurement of ear thickness: No signs of irritation indicated by the increase in mean ear thickness were noted.

Body weight: There was no indication of an effect of treatment on body weight gain. 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.

Table 7.4.1/1 : Individual Disintegrations per Minute and Stimulation Indices

Treatment Group

Animal Number

dpm/ Animala

Mean dpm/Animal (Standard Deviation)

Stimulation Indexb

Result

Vehicle acetone/olive oil 4:1

1-1

2108.49

2217.80 (±203.05)

na

na

1-2

2298.16

1-3

2535.86

1-4

2115.36

1-5

2031.12

Test Item 25% v/v in acetone/olive oil 4:1

2-1

7472.53

6870.78** (±720.83)

3.10

Positive

2-2

6342.71

2-3

6277.67

2-4

7819.96

2-5

6441.01

Test Item 50% v/v in acetone/olive oil 4:1

3-1

14710.36

19637.09** (±5036.07)

8.85

Positive

3-2

25912.11

3-3

18322.54

3-4

23867.04

3-5

15373.41

Test Item 100%

4-1

34319.39

39532.59** (±7449.68)

17.83

Positive

4-2

46971.60

4-3

29440.18

4-4

45292.91

4-5

41638.87

Positive Control Item

25% v/v in acetone/olive oil 4:1

5-1

20309.44

12922.88** (±7257.76)

5.83

Positive

5-2

21265.82

5-3

7030.16

5-4

9313.81

5-5

6695.15

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal 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 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test material is classified as a skin sensitizer 1B according to the annex VI of the Regulation EC No. 1272/2008 (CLP).
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

The study comprised three treated groups, each comprising five female mice receiving test item at concentrations of 25, 50% v/v or the undiluted test item. Similarly constituted groups received the vehicle 4:1 v/v acetone: olive oil or positive control substance (25% v/v α-Hexylcinnamaldehyde). The mice were treated by daily application of 25mL of the appropriate concentration or control (vehicle or positive), to the dorsal surface of both ears for three consecutive days.The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. 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 (Stimulation Index).

The SI obtained for 25, 50% v/v and for the undiluted test item were 3.10, 8.85 and 17.83 respectively which indicates that test item showed the potential to induce skin sensitization. The EC3 value was calculated to be 24.7% v/v. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 25 and 50 v/v, nor for the undiluted test item.

 

The SI for the positive control substance α-Hexylcinnamaldehyde was 5.83, which demonstrates the validity of this study.

 

Under the test conditions, test material is classified as a skin sensitizer 1B in the Local Lymph Node Assay according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

 

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.