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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:
From October 2016 to September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
the actual relative humidity range was 23-84 % instead of 30-70 % and the actual temperature range was 16.8- 24.8°C instead of 22 ± 3°C. This deviation is considered not to adversely affect the results or integrity of the study
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
the actual relative humidity range was 23-84 % instead of 30-70 % and the actual temperature range was 16.8- 24.8°C instead of 22 ± 3°C. This deviation is considered not to adversely affect the results or integrity of the study
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
the actual relative humidity range was 23-84 % instead of 30-70 % and the actual temperature range was 16.8- 24.8°C instead of 22 ± 3°C. This deviation is considered not to adversely affect the results or integrity of the study
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Solvent N1
- Physical state: liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 24/02/2016
- Expiration date of the batch: July 2021
- Purity test date: 75.6 % (w/v) N1 SOLVENT

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature 15-25°C, below 70 RH%
- Solubility and stability of the test substance in the solvent/vehicle: Stability not checked because of the character and the short period of study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulations were prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd. Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 11 weeks old (age-matched, within one week)
- Weight at study initiation: 21.1 – 22.8 g
- Housing: Individual caging / mice were provided with glass tunnel-tubes (Cage type: Type II. polypropylene / polycarbonate)
- Diet (e.g. ad libitum): Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” ad libitum.
- Water (e.g. ad libitum): Animals received tap water from the municipal supply from 500 mL bottle, ad libitum.
- Acclimation period: 28 days
- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.8 - 24.8°C
- Humidity (%): 23 - 84 %
- 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.
- IN-LIFE DATES: From: 09 November 2016 To: 16 November 2016

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
Preliminary Irritation/Toxicity Test :concentrations of 100 % (undiluted) and 50 % (w/v) in MEK.

Main test : concentrations of 100 % (undiluted), 50 % (w/v) and 25 % (w/v) in MEK.
No. of animals per dose:
Preliminary Irritation/Toxicity Test : 2 animals/dose

Main test : 5 animals/dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: DMF was selected as vehicle in the preliminary experiment. In the preliminary experiment the test material (65.4% dilution in water), and a 50% dilution of this in DMF were evaluated (2 animals/dose).
- Irritation: no signs of local irritation were observed in the preliminary test.
- Systemic toxicity: no systemic toxicity were observed in the preliminary test
- Ear thickness measurements: Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. Slightly increased ear thickness values were detected in some cases on Days 3 and/or Day 6; but the mean values were well below the irritant response.
- Erythema scores: No erythema was observed for any experimental animals in the preliminary experiment.


MAIN STUDY:

ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.
- Criteria used to consider a positive response:
Stimulation index (SI = DPN (disintegrations per minute divided by the number of lymph nodes) value of a treated group divided by the DPN value of the negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.
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 (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Based on the results of the preliminary study, 100 % (undiluted) dose was selected as top dose for the main test. Two additional groups of 5 animals each were treated at the test concentration of 50% and 25% respectively in MEK.

Topical application:
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. As the test item is expected to be volatile,the application was performed within 15 minutes after formulation. 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.

OBSERVATIONS:
1) Clinical 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 at least twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

2) Measurement of Body Weight:
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

3) Ear Thickness Measurements:
In the preliminary experiment, both ears of each mouse were observed for erythema and scored according to the guideline's criteria. 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 in the preliminary experiment. Additional quantification of the ear thickness in the preliminary experiment was also performed by ear punch weight determination on Day 6 after the euthanasia of the experimental animals.

PROLIFERATION ASSAY:
1) Injection of Tritiated Thymidine (3HTdR):
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

2) Removal and Preparation of Draining Auricular Lymph Nodes:
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

3) Preparation of Single Cell Suspension of Lymph Node Cells:
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

4) Determination of Incorporated 3HTdR:
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed.
Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

EVALUATION OF THE RESULTS:
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistics performed.

Results and discussion

Positive control results:
The positive control substance was examined at a concentration of 25 % in the relevant vehicle (MEK) using CBA/CaOlaHsd mice
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. lymphoproliferative response in line with historic positive control data (stimulation index value of 16.6) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
< 3
Parameter:
SI
Value:
0.9
Test group / Remarks:
100 % (undiluted)
Parameter:
SI
Value:
0.9
Test group / Remarks:
50% (w/v) in MEK
Parameter:
SI
Value:
0.8
Test group / Remarks:
25% (w/v) in MEK
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
Theappearance of the lymph nodes was normal in the negative control group and in all the test item treated dose groups. Larger than normal lymph nodes were observed in the positive
control group.

DETAILS ON STIMULATION INDEX CALCULATION:
The stimulation index values were 0.9, 0.9 and 0.8 at concentrations of 100 % (undiluted), 50 and 25 % (w/v), respectively. (see table 1)

EC3 CALCULATION: No calculated (SI < 3 in all concentrations tested)

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity was observed during the main test. There were no visual indications of any irritancy at the site of application.

BODY WEIGHTS:
No marked body weight loss (≥5%) was observed on the mean body weight change; however, the body weight loss was more than 5% for one animal in the 100 % (undiluted),
dose group and for two animals in the negative control group.

Any other information on results incl. tables

Table 1: DPM, DPN and Stimulation Index Values for all Groups:

Test Group Name

Identity

Number

Measured

DPM

DPM

Number of

lymph

nodes

DPN

Group

DPN

 

Stimulation

Index

Background

(5 % (w/v) TCA)

-

30

-

-

-

-

-

 

30

-

-

-

-

-

Negative (vehicle)

control

(MEK)

1

259

229

2

114.5

120.2

1.0

2

191

161

2

80.5

3

343

313

2

156.5

4

350

320

2

160.0

5

209

179

2

89.5

N1 SOLVENT

100 % (undiluted)

6

281

251

2

125.5

110.65

0.9

7

138

108

2

54.0

8

251

221

2

110.5

9

307

277

2

138.5

10

279

249

2

124.5

N1 SOLVENT

50 % (w/v)

in MEK

11

349

319

2

159.5

106.3

0.9

12

354

224

2

112.0

13

168

138

2

69.0

14

210

180

2

90.0

15

232

202

2

101.0

N1 SOLVENT

25 % (w/v)

in MEK

16

237

207

2

103.5

95.7

0.8

17

216

186

2

93.0

18

231

201

2

100.5

19

235

205

2

102.5

20

188

158

2

79.0

Positive control

(25 % HCA in

MEK)

21

4225

4195

2

2097.5

1990.7

16.6

22

3155

3125

2

1562.5

23

3620

3590

2

1795.0

24

3830

3800

2

1900.0

25

5227

5197

2

2598.5

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the conditions of the present assay, N1 SOLVENT, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay..
Executive summary:

The aim of this study was to determine the skin sensitisation potential of N1 SOLVENT following dermal exposure. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline no. 429 [1], the test item was tested for formulation compatibility in Methyl ethyl ketone (abbreviated as MEK). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted).

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100 % (undiluted) and 50 % (w/v) in MEK. Based on the observations recorded in the preliminary test, the 100 % (undiluted) was selected as top dose for the main test.

In the main assay, twenty-five female CBA/CaOlaHsd mice were allocated to five groups of five animals each:

- three groups received N1 SOLVENT (formulated in MEK) at 100 % (undiluted), 50 and 25 % (w/v) concentrations,

- the negative control group received the vehicle (MEK),

- the positive control group received 25 % (w/v) HCA (dissolved in MEK).

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. 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. There were no indications of any irritancy at the site of application.

The stimulation index values were 0.9, 0.9 and 0.8 at concentrations of 100 % (undiluted), 50 and 25 % (w/v), respectively.

All the validity criteria were met.

In conclusion, under the conditions of the present assay, N1 SOLVENT, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.