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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 January 2015 - 24 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 429. GLP study.

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
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)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
EC Number:
309-913-1
EC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
Cas Number:
101357-16-8
Molecular formula:
Not applicable. Multiconstituent substance.
IUPAC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Solvent Black 5 (Nigrosine Alcohol BXL)
- Physical state: Black powder
- Analytical purity: 102.0% (measured value)
- Lot/batch No.: 104/14
- Expiration date of the lot/batch: 17 October 2019

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories S.r.l. San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Age at study initiation: 9 weeks old
- Weight at study initiation: 17.4-19.3 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage type: Type II. polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ad libitum, ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: al least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-24.8°C
- Humidity (%): 31-69 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
(DMF)
Concentration:
10, 5, 2.5%
No. of animals per dose:
4 animals per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility (Preliminary Compatibility Test): The following standard OECD vehicles were assessed: Acetone : Olive oil 4:1 (v:v) mixture (AOO), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200 (1% Pluronic). The 100, 50 and 25% (w/v) formulations were not achievable using any of these vehicles. The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be DMF. The highest achievable concentration for the test based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 10% (w/v).
- Preliminary Irritation/Toxicity Test: 2 animals per dose were exposed to test item concentrations of 10 and 5 % (w/v) in DMF. The experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. No mortality or clinical signs of systemic toxicity were observed. No marked body weight loss (>5%) was observed except of one animal in the 10% (w/v) dose group, but was considered as animal variability. Test item precipitate was observed on the ears of the animals in both dose groups on Days 1-3. Increased ear thickness values were detected in some cases; but the resulted data were below the limit of positivity in all cases. The revealing ear punch weights were within the historical control range. There was no indication of irritation at the site of application based on the visual assessment. The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
*That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in
control mice, as indicated by the stimulation index.
*The data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On day 6, each mouse received an injection of 250µl of sterile PBS (phosphate buffered saline) containing approx. 20 µCi of 3HTdR. Five hours later, the mice were euthanized and the auricular lymph nodes were extracted from the animals. A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and the samples were prepared to be examined in a β-scintillation counter.

OBSERVATIONS:
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection).

EVALUATION OF RESULTS:
Radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes and corrected with the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes). Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Test item 10 % (w/v) in DMF: 3641 DPM Test item 5 % (w/v) in DMF: 3711 DPM Test item 2.5 % (w/v) in DMF: 4127 DPM
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
at concentration of 10% (w/v)
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
at concentration of 5%.
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
at concentration of 2.5%.

Any other information on results incl. tables

Clinical observation:

No mortality or signs of systemic toxicity were observed during the study. Test item precipitate (minimal amount) was observed on the ears of the animals in all test item treated group on Days 1 -6. There were no indications of any irritancy at the site of application.

Body weight:

No treatment related body weight loss was detected in any test item treated animals. Increased body weights compared to the negative control group were detected in the 10% dose group.

Proliferation assay:

Test Group Name

Measured DPM / group

DPM

Number
lymph nodes

DPN

Stimulation Index

Background (5 % (w/v) TCA)

33/59

-

-

-

-

(-) control (DMF)

2034

1988.0

8

248.5

1.0

Test item 10 % (w/v) in DMF

3641

3595.0

8

449.4

1.8

Test item 5 % (w/v) in DMF

3711

3665.0

8

458.1

1.8

Test item 2.5 % (w/v) in DMF

4127

4081.0

8

510.1

2.1

(+) control (25 % (w/v) HCA in DMF

18798

18752.0

8

2344.0

9.4

The appearance of the lymph nodes was normal in the negative (vehicle) control group and in all test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item showed to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The stimulation index values were 1.8, 1.8 and 2.1 at concentrations of 10, 5 and 2.5 % (w/v), respectively.
Executive summary:

The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42 (GLP study). Based on the results of the Preliminary Compatibility Test, the test item was formulated in  N,N-Dimethylformamide (abbreviated as DMF) at a highest achievable concentration of 10 % (w/v). The Preliminary Irritation / Toxicity Test was performed in CBA/CaOlaHsd mice using two doses: 10 and 5 % (w/v) in DMF. The 10 % (w/v) was selected as top dose for the main test. Twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each in the main test. Three groups received the test item at 10, 5 and 2.5 % (w/v) concentrations, the negative control group received the vehicle (DMF) and the positive control group received 25 % (w/v) HCA (dissolved in DMF). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or signs of systemic toxicity were observed during the study. No treatment related body weight loss was observed in the test item treated animals. Test item precipitate (minimal amount) was observed on the ears of the animals in all test item treated groups on Days 1-6. There were no indications of any irritancy at the site of application. The stimulation index values were 1.8, 1.8 and 2.1 at concentrations of 10, 5 and 2.5% (w/v), respectively. Since there were no confounding effects of irritation or significant systemic toxicity at any examined concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation index values observed at the concentrations of 10, 5 and 2.5% (w/v) were below the threshold limit of 3, thus they were considered to be good evidence that the test item is not a sensitizer. The size of lymph nodes was in good correlation with this conclusion.

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