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EC number: 431-480-3 | CAS number: -
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 Apr 2018 to 15 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was performed according to the OECD guideline No.429 (July 2010) and in compliance with the GLP.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- OECD Guideline 429. Skin Sensitization: Local Lymph Node Assay, July 2010.
EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay, July 2012. - Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Test material form:
- solid: particulate/powder
Constituent 1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA:J
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS:
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 10 weeks old) were selected.
Weight at the Initiation of Dosing: 18.4 to 23.5 g.
ENVIRONMENTAL CONDITIONS:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing
Target temperatures of 18 to 24°C
relative target humidity of 40 to 70%
Daily mean temperature: 22 to 23°C
Mean relative humidity of 41 to 51%
A 12 hour light/12 hour dark cycle was maintained.
Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Study design: in vivo (LLNA)
- Vehicle:
- dimethylformamide
- Concentration:
- For the preliminary test the concentrations were 5,10, 25, and 50% of the test item.
For the main test the concentrations were 0, 2, 5 and 10% of the test item. - No. of animals per dose:
- For the preliminary test: 4 females/dose
For the main test: 5 females/dose, 5 females for the negative control and 5 females for the positive control
see details in table results - Details on study design:
- Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Initially, two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (5% and 10%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 9-11 weeks (at initiation of treatment) the application method may have been different (see tables) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.
Main Study:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
Induction - Days 1, 2 and 3:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
Irritation:
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). According to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
Erythema and eschar formation:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4
ANALYSIS:
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3) (reference 1). - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
Results and discussion
- Positive control results:
- In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and stimulation index exceeding the thresold value of 3 (SI=3.5 +/-0.6%) was noted.
The study is therefore considered as valid
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- ca. 1.8
- Variability:
- +/-0.2
- Test group / Remarks:
- 10%
- Key result
- Parameter:
- SI
- Value:
- 2.2
- Variability:
- +/- 0.3
- Test group / Remarks:
- 5 %
- Key result
- Parameter:
- SI
- Value:
- 1.4
- Variability:
- +/- 0.1
- Test group / Remarks:
- 2%
Any other information on results incl. tables
Main study -results
Skin Reactions / Irritation
The very slight erythema of the ears as shown by three animals treated at 10% on Day 3 and the scaliness as noted for animals of all groups between Days 4 and 6 were considered not to have a toxicologically significant effect on the activity of the nodes.
Light yellow test item remnants were present on the dorsal surface of the ears all test item treated animals throughout the observation period, which did not hamper scoring of the skin reactions.
Systemic toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period
Macroscopic Examination of the Lymph Nodes and Surrounding Area
The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 5% and three animals treated at 10% which were considered to be enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Main Study: Body Weights and Skin Reactions
group | TS1 (%) | animal | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | ||||||||
bw (g)2 | erythema3 | erythema | erythema | erythema | erythema | erythema | bw (g) | |||||||||
left | right | left | right | left | right | left | right | left | right | left | right | |||||
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1 | 0 | 1 | 22.4 | 0 | 0 | 0 | 0 | 0 | 0 | 0s | 0s | 0s | 0s | 0 | 0 | 24.1 |
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| 2 | 19.9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 21.5 |
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| 3 | 19.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0s | 0s | 0s | 0 | 0 | 20.6 |
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| 4 | 23.5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 24.5 |
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| 5 | 20.7 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0s | 0 | 0 | 0 | 23.5 |
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2 | 2 | 6 | 20.6 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0 | 0 | 24.6 |
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| 7 | 19.3 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0f | 0s | 0 | 22.1 |
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| 8 | 18.9 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 22.3 |
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| 9 | 19.6 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0fs | 0 | 0s | 21.7 |
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| 10 | 18.4 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 21.6 |
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3 | 5 | 11 | 20.9 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 21.9 |
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| 12 | 21.0 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0s | 19.5 |
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| 13 | 19.0 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0fs | 0f | 0 | 22.5 |
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| 14 | 21.5 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0f | 0fs | 0fs | 0s | 0s | 23.5 |
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| 15 | 21.5 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0fs | 0s | 0s | 24.0 |
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4 | 10 | 16 | 22.9 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0f | 0fs | 0fs | 23.1 |
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| 17 | 21.5 | 0f | 0f | 0f | 0f | 1f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 22.5 |
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| 18 | 20.5 | 0f | 0f | 0f | 0f | 1f | 1f | 0f | 0f | 0f | 0f | 0fs | 0fs | 21.1 |
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| 19 | 21.4 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 22.7 |
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| 20 | 21.2 | 0f | 0f | 0f | 0f | 1f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 23.3 |
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f. Light yellow staining of the dorsal surface of the ears by test item remnants which did not hamper the scoring
of the ears s.Scaliness
1 TS = test item (% w/w).
2 Body weight (grams).
3 Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):
0 = No erythema
1 = Very slight erythema (barely perceptible)
Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)
group | TS1 (%) | animal | Size nodes2 | DPM3/ animal | mean DPM ± SEM4 | mean SI ± SEM | |||||
left | right | ||||||||||
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1 | 0 | 1 | n | n | 482 | 538 | ± | 94 | 1.0 | ± | 0.2 |
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| 2 | n | n | 660 | ||||||
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| 3 | n | n | 223 | ||||||
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| 4 | n | n | 548 | ||||||
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| 5 | n | n | 778 | ||||||
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2 | 2 | 6 | n | n | 475 | 727 | ± | 76 | 1.4 | ± | 0.1 |
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| 7 | n | n | 806 | ||||||
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| 8 | n | n | 877 | ||||||
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| 9 | n | n | 630 | ||||||
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| 10 | n | n | 849 | ||||||
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3 | 5 | 11 | n | n | 541 | 1166 | ± | 185 | 2.2 | ± | 0.3 |
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| 12 | n | n | 1584 | ||||||
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| 13 | n | n | 1142 | ||||||
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| 14 | + | n | 1494 | ||||||
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| 15 | n | n | 1071 | ||||||
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4 | 10 | 16 | + | + | 1152 | 982 | ± | 131 | 1.8 | ± | 0.2 |
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| 17 | n | + | 1395 | ||||||
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| 18 | n | n | 919 | ||||||
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| 19 | n | n | 757 | ||||||
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| 20 | + | n | 686 | ||||||
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1 TS = test item (% w/w).
2 Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).
3 DPM= Disintegrations per minute.
4 SEM = Standard Error of the Mean.
Pre-screen Test - Results
At a 50% and 25% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and therefore this concentration did not meet the selection criteria.
At a 10% and 5% test item concentration, no signs of systemic toxicity were noted and no significant irritation was observed and therefore the 10% concentration was selected as highest concentration for the main study
| Table 1 Pre-Screen Test: Body Weights and Skin Reactions | ||||||||||||||||
| TS1 | (%) | animal | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | ||||||||
| bw | erythema3 | erythema | erythema | erythema | erythema | erythema | bw | |||||||||
| (g)2 | left | right | left | right | left | right | left | right | left | right | left | right | g) | |||
| 504 | 3 | 23,6 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0s | 0s | 25,1 | |
| 4 | 22,2 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0s | 0 | 22,4 | ||
| 25 | 1 | 24,7 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0s | 0s | 25,8 | |
| 2 | 22,8 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0 | 0s | 23,6 | ||
| 10 | 7 | 21 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0s | 0s | 21,6 | |
| 8 | 21,3 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0s | 22,2 | ||
| 5 | 5 | 21 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0f | 0f | 20,8 | ||
| 6 | 18,4 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0s | 0s | 19,8 | ||
| f.White staining of the dorsal surface of the ears by test item remnants which did not hamper the scoring of the ears | ||||||||||||||||
| s.Scaliness | ||||||||||||||||
| 1TS = test item (% w/w). | ||||||||||||||||
| 2Body weight (grams). | ||||||||||||||||
| 3Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear): 0 = No erythema | ||||||||||||||||
| 4Applied using spatula instead of using a pipette. | ||||||||||||||||
| Table 2 Pre-Screen Test: Ear Thickness Measurements | ||||||||||||
| TS1 | (%) | animal | Day 1 | Day 3 | Day 6 | |||||||
| left | right | left | right | right | left | right | left | right | ||||
| (mm) | (mm) | (mm) | %2 | (mm) | %2 | (mm) | %2 | (mm) | %2 | |||
| 50 | 3 | 0,22 | 0,215 | 0,28 | 27 | 0,285 | 33 | 0,23 | 5 | 0,225 | 5 | |
| 4 | 0,22 | 0,225 | 0,285 | 30 | 0,29 | 29 | 0,225 | 2 | 0,225 | 0 | ||
| 25 | 1 | 0,22 | 0,225 | 0,29 | 32 | 0,3 | 33 | 0,22 | 0 | 0,225 | 0 | |
| 2 | 0,225 | 0,225 | 0,29 | 29 | 0,295 | 31 | 0,225 | 0 | 0,23 | 2 | ||
| 10 | 7 | 0,235 | 0,23 | 0,24 | 2 | 0,24 | 4 | 0,23 | -2 | 0,225 | -2 | |
| 8 | 0,225 | 0,23 | 0,24 | 7 | 0,24 | 4 | 0,22 | -2 | 0,225 | -2 | ||
| 5 | 5 | 0,225 | 0,23 | 0,24 | 7 | 0,235 | 2 | 0,22 | -2 | 0,225 | -2 | |
| 6 | 0,225 | 0,225 | 0,24 | 7 | 0,23 | 2 | 0,22 | -2 | 0,23 | -2 | ||
| Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters. | ||||||||||||
| 1TS = test item (% w/w). | ||||||||||||
| 2Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for use in the main study. | ||||||||||||
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 3).
Based on these results,test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments). - Executive summary:
The objective of this study was to evaluate whether test item induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this study plan.
The study was carried out based on the guidelines described in:
· OECD, Section 4, Health Effects, No.429 (2010),
· EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"
· EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.
Test item concentrations selected for the main study were based on the results of a pre-screen test. The 50% and 25% test item concentration did not meet the selection criteria. The 10% and 5% test item concentration did meet the selection criteria and therefore the 10% concentration was selected as highest concentration for the main study.
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-dimethylformamide). Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.
The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 5% and three animals treated at 10% which were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 727, 1166 and 982 DPM, respectively. The mean DPM/animal value for the vehicle control group was 538 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 1.4, 2.2 and 1.8, respectively.
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