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Toxicological information

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:
05 April 2018 - 22 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: housed in suspended solid floor polypropylene cages furnished with softwood wood flakes
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK; ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
preliminary test: 50%, 25%, 10% and 5% v/v in acetone/olive oil 4:1
main test: 10%, 5% or 2.5% v/v in acetone/olive oil 4:1
No. of animals per dose:
preliminary test: 1
main test: 5
Details on study design:
PRE-SCREEN TESTS:
- Systemic toxicity: The animals treated with the test item at concentrations of 50% and 25%% v/v in acetone/olive oil 4:1 were humanely killed, on Day 3 or Day 5, due to the occurrence of clinical signs of toxicity that were considered to approach the moderate severity limit set forth in the UK Home Office Project License. No signs of toxicity or excessive irritation were noted ion the animals treated at concentrations of 5% and 10% v/v in acetone/olive oil 4:1.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
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 test item will be regarded as a sensitizer 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 sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Administration:
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.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1, was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item, vehicle control item or positive control item (Day 6) all mice were injected via the tail vein with 0.25 mL (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.

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.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures:
Termination: 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 processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
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 labeled 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 C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by  scintillation counting. The "Poly Q™" 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 the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

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.

Results and discussion

Positive control results:
S.I. = 4.29 (positive)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
21.25
Test group / Remarks:
test item: 2.5% v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
35.7
Test group / Remarks:
test item: 5% v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
47.06
Test group / Remarks:
test item: 10% v/v in acetone/olive oil 4:1
Cellular proliferation data / Observations:
EC3 CALCULATION
An EC3 value could not be calculated, since all tested concentrations resulted in an S.I. >3

CLINICAL OBSERVATIONS:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Individual Disintegrations per Minute and Stimulation Index

Treatment Group

Animal Number

dpm/
Animala

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle
acetone/olive oil 4:1

1-1

578.14

646.71
(±166.91)

na

na

1-2

886.46

1-3

620.01

1-4

436.45

1-5

712.51

Test Item
2.5v/vin
acetone/olive oil 4:1

2-1

13285.92

13743.18**
(±1651.86)

21.25

Positive

2-2

12205.57

2-3

13811.24

2-4

16506.63

2-5

12906.54

Test Item
5v/vin
acetone/olive oil 4:1

3-1

29351.63

23084.47**
(±4665.87)

35.70

Positive

3-2

20994.83

3-3

23902.66

3-4

24478.24

3-5

16694.99

Test Item
10v/vin
acetone/olive oil 4:1

4-1

29032.68

30435.92**
(±3109.43)

47.06

Positive

4-2

31591.13

4-3

30843.87

4-4

26161.23

4-5

34550.67

Positive Control Item
25% v/vin
acetone/olive oil 4:1

5-1

4860.74

2771.32****
(±1213.29)

4.29

Positive

5-2

2295.43

5-3

1722.89

5-4

2352.30

5-5

2625.25

  


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 control group p<0.01

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
CGE-PMDA adduct was considered to be a sensitizer under the conditions of the test.
Executive summary:

A Local Lymph Node Assay according to OECD Guideline 429 was performed to assess the skin sensitization potential of CGE-PMDA adduct in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following preliminary screening tests in which no clinical signs of toxicity were noted at a concentration of 10% v/v, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five females, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 10%, 5% or 2.5% v/v. A further group of five animals were treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment Group

Concentration

Stimulation Index

Result

Test Item

2.5% v/v in
acetone/olive oil 4:1

21.25

Positive

5% v/v in
acetone/olive oil 4:1

35.70

Positive

10% v/v in
acetone/olive oil 4:1

47.06

Positive

Positive Control Item

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

4.29

Positive

 

CGE-PMDA adduct was considered to be a sensitizer under the conditions of the test.

No EC3 could be calculated, since all S.I. values were > 3. Nevertheless, based on the results it can be concluded that the EC3 will be <<3. Thus, the substance is classified as Category 1A.