Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 June 2015 to 29 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: Slightly yellow powder with lumps
- Storage conditions of test material: At room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/J strain, inbred, SPF-Quality
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 21 to 24 g. Body weight variation was within ± 20 % of the sex mean.
- Housing: During acclimation animals were group housed in labelled cages (height 18 cm) containing sterilised sawdust as bedding material; paper and shelters were supplied as cage-enrichment. After acclimation animals were group housed in cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: 10 June 2015
To: 29 June 2015

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25 and 50 % test material w/w
No. of animals per dose:
5 female animals per dose
Details on study design:
PRE-SCREEN TEST
A pre-screen test was conducted in order to select the highest test material 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.
Two test material concentrations were tested; 50 and 25 %. The highest concentration was the maximum concentration as required in the test guidelines (50 % for solids).
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) 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 labelled cages (height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge 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 material concentration per group. The highest test material concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

INDUCTION (Days 1, 2 and 3)
The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, 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 material.

EXCISION OF THE NODES (Day 6)
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of ³H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20 %. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

TISSUE PROCESSING FOR RADIOACTIVITY (Day 6)
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS (Day 7)
Precipitates were recovered by centrifugation, re-suspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes, whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

OBSERVATIONS
- Mortality/Viability: Twice daily
- Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy)
- Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing)
- Irritation: 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.
Grading Irritation Reactions (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
- Necropsy: No necropsy for gross macroscopic examination was performed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value (the estimated test substance concentration that will give an SI =3) was determined, using linear interpolation (Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl. Toxicol. 1999; 19:261-266.

Results and discussion

Positive control results:
A reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v) was performed approximately 2 months prior to the study, using the same materials, animal supplier, animal strain and essential procedures as for the main test. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.
Concentrations used for this study were 5, 10 and 25 % in Acetone/Olive oil (4:1 v/v); the SI values calculated were 1.7, 3.0 and 9.1, respectively. An EC3 value of 10 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range. Based on the results, it was concluded that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI values calculated for the test material concentrations 10, 25 and 50 % were 1.7, 3.3 and 4.6, respectively. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI =3) of 22.2 % was calculated.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test material concentrations 10, 25 and 50 % were 1155, 2289 and 3174 DPM, respectively. The mean DPM/animal value for the vehicle control group was 692 DPM.

Any other information on results incl. tables

Pre-Screen Test

No irritation and no signs of systemic toxicity were observed in any of the animals examined. White staining of test material remnants on the dorsal surface of the ears of all animals on Days 2 and 3 did not hamper scoring for erythema.

Variations in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values.

 

Main Study

Skin reactions / Irritation

No irritation of the ears was observed in any of the animals examined. White staining of test material remnants on the dorsal surface of the ears of one animal treated at 10 % and all animals treated at 25 and 50 % did not hamper scoring for erythema.

 

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.

 

Macroscopy of the auricular lymph nodes and surrounding area

All auricular lymph nodes of the animals of the 10 % and control groups were considered normal in size. The auricular lymph nodes of two animals treated at 25 % and all animals treated at 50 % appeared larger in size.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Table 1: Relative size of lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

Test material (% w/w)

Animal

Number

Size nodes*

DPM¹/ animal

Mean DPM ± SEM³

 

Mean SI ± SEM

Left

Right

0

1

n

n

542

692 ± 132

1.0 ± 0.3

2

n

n

1215

3

n

n

622

4

n

n

581

5

n

n

499

10

6

n

n

1143

1155 ± 146

1.7 ± 0.4

7

n

n

1226

8

n

n

814

9

n

n

932

10

n

n

1661

25

16

+

+

1466

2289 ± 388

3.3 ± 0.8

17

n

n

2587

18

+

+

2403

19

n

n

1459

20

n

n

3530

50

11

+

+

2664

3174 ± 576

4.6 ± 1.2

12

+

+

4552

13

+

+

4208

14

+

+

1328

15

+

+

3119

*Relative size auricular lymph nodes (+,++ or +++: degree of enlargement, n: considered to be normal)

¹DPM = Disintegrations per minute

³SEM = Standard Error of the Mean

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study, the results indicate that the test material could elicit a SI ≥ 3. The test material is therefore considered to be a sensitiser.
Executive summary:

An assessment of the potential for the test material to cause skin sensitisation was made in the mouse in a Local Lymph Node Assay (LLNA) study carried out in accordance with the standardised guidelines OECD 429, EU Method B.42 and US EPA OPPTS 870.2600 under GLP conditions.

The test material concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 10, 25 or 50 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (N,N-Dimethyl formamide).

Three days after the last exposure, all animals were injected with ³H-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.

No irritation of the ears was observed in any of the animals examined. White staining of test material remnants on the dorsal surface of the ears of one animal treated at 10 % and all animals treated at 25 and 50 % did not hamper scoring for erythema.

 All auricular lymph nodes of the animals of the 10 % and control groups were considered normal in size. The auricular lymph nodes of two animals treated at 25 % and all animals treated at 50 % appeared larger in size. 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 material concentrations 10, 25 and 50 % were 1155, 2289 and 3174 DPM, respectively. The mean DPM/animal value for the vehicle control group was 692 DPM. The SI values calculated for the material concentrations 10, 25 and 50 % were 1.7, 3.3 and 4.6, respectively.

These results indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI =3) of 22.2 % was calculated.

Under the conditions of this study, the test material is considered to be a sensitiser.