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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:
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
Report Date:
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
Remarks:
Clear colourless to slighty yellowish liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. 99270
- Expiration date of the lot/batch: 6.11.2019
- Purity test date: not detailed

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: I125iododeoxyuridine
- Specific activity:14-150 µCi
- Locations of the label: I
- Expiration date of radiochemical substance: not detailed

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20±5°C)
- Stability under test conditions: not stated
- Solubility and stability of the test substance in the solvent/vehicle: soluble and homogeneous solution in the vehicle
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Velaz
- 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: 16.43 to 22.92g
- 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 test : 100% and 25%
main test : 10, 5 and 2.5%
No. of animals per dose:
Pre test : 2 animals per condition 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. Erythema scores are shown in table 1. 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 an SI of 6.22 and an increase of lymph node weight.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
6.22
Test group / Remarks:
Positive Control
Key result
Parameter:
SI
Value:
5.65
Test group / Remarks:
Trihexylamine 2.5%
Key result
Parameter:
SI
Value:
15.11
Test group / Remarks:
Trihexylamine 5%
Key result
Parameter:
SI
Value:
21.44
Test group / Remarks:
Trihexylamine 10%
Key result
Parameter:
EC3
Value:
2.06
Test group / Remarks:
Trihexylamine
Cellular proliferation data / Observations:
PRESCREEN TEST
Pre-screen test - concentration of 100%
Trihexylamine administered at concentration of 100%, caused death of both mice within 24 hours.
Pre-screen test - concentration of 25%
Administration of Trihexylamine at concentration of 25% caused apathy, weak intake of food, increase of mean ear thickness and decrease of mean body weight

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 0.0894g for 2.5% concentration, 0.1116g for 5% concentration and 0.1g for 10% concentration of tested item. The lymph node weight of control group and positive control group were 0.0356g and 0.0778g, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The DPM values for the three treated groups were 7711 (2.5%), 20629 (5%) and 29263 (10%), respectively. The SI values for the three treated groups were 5.65 (2.5%), 15.11 (5%) and 21.44 (10%), respectively. Because there were no data points that fall below SI = 3, a log-linear extrapolation was applied in which the SI values of the two lowest test concentrations (2.5 and 5 %) were used for calculation of the EC3 value. The EC3 value was calculated to be 2.06%.

EC3 CALCULATION
Calculations were detailed in the section "Details on the study design"

CLINICAL OBSERVATIONS:
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.

BODY WEIGHTS
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. A minor increase of body weight was observed at all used concentrations. The increase was not dose dependent and without statistical significance.

Any other information on results incl. tables

Table 1. Clinical Observations in pre screen test with test substance 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

0

-

-

-

-

-

-

-

-

-

-

Ataxia

0

0

-

-

-

-

-

-

-

-

-

-

Intake of food

0

0

-

-

-

-

-

-

-

-

-

-

Intake of water

0

0

-

-

-

-

-

-

-

-

-

-

Tremor

0

0

-

-

-

-

-

-

-

-

-

-

Change in grooming activity

0

0

-

-

-

-

-

-

-

-

-

-

Pilo-erection

0

0

-

-

-

-

-

-

-

-

-

-

- notexamined        0 no effect        

 

Table 5. Clinical Observationsinpre screentest with test substance at 25%

 

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

0

0

0

+

+

+

+

++

+

++

+

Ataxia

0

0

0

0

0

0

0

0

0

0

0

0

Intake of food

0

0

0

0

Weak

Weak

Weak

Weak

Weak

Weak

Weak

Weak

Intake of water

0

0

0

0

Weak

Weak

Weak

Weak

Weak

Weak

Weak

Weak

Tremor

0

0

0

0

0

0

0

0

0

0

0

0

Change in grooming activity

0

0

0

0

0

0

0

0

0

0

0

0

Pilo-erection

0

0

0

0

0

0

0

0

0

0

0

0

       0 noeffect        + weak effect         ++ moderate effect         

 

Table 3.Erythema score inpre screentest with test substance at 25%

Mouse no.

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

1

0

0

1

1

1

2

2

0

0

1

1

1

2

 

Table 4.Lymph node weight, DPM, SI, EC3 values in main test

 

Lymph node

Number of

 

 

 

 

weight (g)

lymph nodes

DPM

SI

EC3 (%)

Control

0.0356

10

1365

-

 

2.06

Positive Control

0.0778

10

7210

6.22*

Trihexylamine2.5%

0.0894

10

7711

5.65

Trihexylamine  5%

0.1116

10

20629

15.11

Trihexylamine10%

0.1424

10

29263

21.44

 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the experimental conditions of the study, the test item trihexylamine induced a calculated EC3 value is 2.06%. 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 and was classified as Sensitizer 1B according to CLP criteria.
Executive summary:

The skin sensitization potential of Trihexylamine 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 25%. Based on the observations recorded in the Pre-screen tests, the concentration of 10 % 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 10, 5 and 2.5%.

All animals survived throughout the test period without showing any clinical signs. In comparison with the control group, the a 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 and therefore log-linear extrapolation was applied in which the two lowest test concentrations from the dose-response curve were used, provided assuming the lowest SI value approaches the value of 3 and that a linear dose-response exists. The calculated EC3 value is 2.06%. 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.

Under the experimental conditions of the study, the test item trihexylamine induced a calculated EC3 value is 2.06%. 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 and was classified as Sensitizer 1B according to CLP criteria.