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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 12 March 2018 to 18 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
batch no. 99671, supplied by the sponsor
- Expiration date of the lot/batch: 9 October 2019
- Purity test date:6 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature 20±5°C
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: The vehicle was selected according to current OECD TG 429, the test item was soluble in the vehicle, therefor a homogeneous solution was obtained
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required amount of the test item (according to the concentration) was mixed with the vehicle shortly before the administration . The preparations were made freshly before each dosing occasion
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: 50% concentration was obtained by mixing of 0.5 mL of test item with 0.5 mL of vehicle
- Final preparation of a solid: the test item was mixed with the vehicle

FORM AS APPLIED IN THE TEST (if different from that of starting material)
in solution in the vehicle

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Velaz, Prague, Czech Republic
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The health condition of animals was examined by a veterinarian before initiation of the study, no more details
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 19.64±0.911g
- Housing: The animals were housed in IVC polycarbonate cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning.
- Diet (e.g. ad libitum): A laboratory food ssniff was served ad libitum, each day approximately at the same time.
- Water (e.g. ad libitum): The animals received tap water for human consumption. Supply of drinking water was unlimited
- Acclimation period: 5 days
- Indication of any skin lesions: no indication of lesion

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 50-60% Relative Humidity
- Air changes (per hr): centrail air conditionning, no more details
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours
- IN-LIFE DATES: From: 15 March 2017 To: 17 April 2018

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre screen test : 100%
Main test : 50, 25, 10%
No. of animals per dose:
Pre test : 2 animals were used
Main test : 5 animals were used per condition
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: the compound is soluble in the vehicle
- Irritation: local irritation was evaluated
- Systemic toxicity: signs of systemic toxicity were evaluated
- Ear thickness measurements: performed
- Erythema scores: performed

The pre-screen test was conducted under the same conditions as the main LLNA study, except the assessment of lymph node proliferation. The undiluted test item was considered as 100% concentration.
Test procedure of Pre-screen test
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.

MAIN STUDY
Schedule :
Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal was recorded. 250µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 10E4 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.

Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs. The animal body weights were measured prior to the first treatment and at the scheduled sacrifice

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation: EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration
d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.
When there are no data points that fall below SI = 3, log-linear extrapolation may be applied in which the two lowest test concentrations from the dose-response curve are used, provided the lowest SI value approaches the value of 3 and that a linear dose-response exists. The equation for EC3 calculation:
EC3 =2^{log2(c)+ (3−d)/(b−d)×[log2(a)−log2(c)]}
a – concentration for next lowest SI above 3, b – next to lowest SI above 3,
c – concentration for lowest SI above 3, d – lowest SI above 3

TREATMENT PREPARATION AND ADMINISTRATION:
The required amount of the test item (according to the concentration) was mixed with the vehicle shortly before the administration (i.e. 50% concentration was obtained by mixing of 0.5 mL of test item with 0.5 mL of vehicle). 25 µL of the test item was applied to the dorsum of each ear. The alpha-Hexyl cinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control induced a SI value calculated at 6.22.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
6.22
Test group / Remarks:
Positive Control
Key result
Parameter:
SI
Value:
7.38
Test group / Remarks:
Triisononylamine 10%
Key result
Parameter:
SI
Value:
12.82
Test group / Remarks:
Triisononylamine 25%
Key result
Parameter:
SI
Value:
13.47
Test group / Remarks:
Triisononylamine 50%
Key result
Parameter:
EC3
Value:
4.78
Test group / Remarks:
Triisononylamine
Cellular proliferation data / Observations:
PRE SCREEN TEST
Triisononylamine administered at concentration of 100%, caused in both mice apathy observed on days 1 and 2; animals did not react on touch and had weak intake of food and water. Third day after application due to strong signs of toxicity, animals were humanely killed. Effect on erythema score and ear thickness was not observed.

Triisononylamine administered at concentration of 50%, did not caused any changes in monitored parameters

CELLULAR PROLIFERATION DATA
In comparison with the control group, an increase of the pooled lymph node weights at all concentrations was observed. The increase was strong and dose dependent. The pooled lymph node weights of treated groups were 0.1157g for 10% concentration, 0.1236g for 25% concentration and 0.1335g for 50% concentration of tested item. The lymph node weight of control group and positive control group were 0.0314g and 0.0778g, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The DPM values for the three treated groups were 11215 (10%), 19493 (25%) and 20478 (50%), respectively. The SI values for the three treated groups were 7.38 (10%), 12.82 (25%) and 13.47 (50%), respectively

EC3 CALCULATION
The EC3 calculation was detailed in section "details on the study design"

CLINICAL OBSERVATIONS:
The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS
The minor increase of body weight was observed at all used concentrations, without statistical significance.

Any other information on results incl. tables

Table 1. Clinical Observations for thePre screentest with test item at 100%

Signs/Day

Day 1

Day 2      

Day 3

Day 4

Day 5

Day 6

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse

2

Mouse 1

Mouse

2

Apathy

+

0

++

+

-

-

-

-

-

-

-

-

Ataxia

-

0

+

+

-

-

-

-

-

-

-

-

Intake of food

Weak

Weak

Weak

Weak

-

-

-

-

-

-

-

-

Intake of water

Weak

Weak

Weak

Weak

-

-

-

-

-

-

-

-

Tremor

0

0

0

0

-

-

-

-

-

-

-

-

Change in grooming activity

0

0

0

0

-

-

-

-

-

-

-

-

Pilo-erection

0

0

0

0

-

-

-

-

-

-

-

-

- notanalysed     0 no effect       + weak effect       ++ moderate effect      +++ strong effect

 

Table 2.Erythema score for thePre screentest with test item at 50%

Mouse no.

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

1

0

0

0

0

0

0

2

0

0

0

0

0

0

 

 

Table 3.The Lymph node weight, DPM, SI, EC3 values for the main test

 

Lymph node

Number of

 

 

 

 

weight (g)

lymph nodes

DPM

SI

EC3 (%)

Control

0.0314

10

1520

-

 

4.78

Positive Control

0.0778

10

7210

6.22*

Triisononylamine10%

0.1157

10

11215

7.38

 Triisononylamine25%

0.1236

10

19493

12.82

 Triisononylamine50%

0.1335

10

20478

13.47

 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the experimental condition of the study, the test substance triisononylamine induced an EC3 value of 4.78% on the LLNA. Hence, according to CLP criteria, the triisononylamine was classified as sensitizer category 1B.
Executive summary:

The skin sensitization potential of Triisononylamine was evaluated by LLNA method, under GLP compliance and according to OECD TG 429 method.

The test item was suspended in Acetone:Olive Oil 4:1 (v/v). The positive control (Alpha-Hexylcinnamic aldehyde) (25%) was dissolved in the same vehicle.

The Pre-screen test was performed using a dose of 100 % and 50%. Based on the observations recorded in the Pre-screen tests, the concentration of 50 % was selected as top dose for the main test.

In the present study, the test item was applied to the dorsum of each ear of five female mice (CBA/Ca) per group over three consecutive days, at three concentrations.

All animals survived throughout the test period without showing any clinical signs. In comparison with the control group, the strong increase in lymph node weight was observed at all used concentrations. The increase of lymph node weight was dose dependent. A similar trend was registered in the evaluation of DPM of the lymph nodes. Calculated SI values in treated groups were higher than 3 at the all used concentrations. The calculated EC3 value is 4.78%. As the SI for some treatment dose group is ≥3 and a clear dose response relationship is observed, the test item is regarded as a potential skin sensitizer.

These results demonstrate that the test item Triisononylamine was a skin sensitizer under the test conditions of this study.

Under the experimental condition of the study, the test substance triisononylamine induced an EC3 value of 4.78% on the LLNA. Hence, according to CLP criteria, the triisononylamine was classified as  sensitizer category 1B.