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Administrative data

Description of key information

The dermal sensitization potential of 1-Benzyl-3 -carbamoyl-pyridinium, chloride (approx. 96% purity) was tested in a murine local lymph node assay (LLNA) conducted according to OECD 429. Neither mortality nor any other adverse effects were observed during the study. Based on the results, the target substance is not a dermal sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-29 to 2017-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
OlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF upon arrival
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 18 -23 g
- Housing: groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): ad libitum; Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): ad libitum; tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: See Remarks
Remarks:
Aqua ad injectionem (AlleMan Pharma, lot no. 605070, expiry date: 04/2019) containing 2% CMC Prior to each dosing carboxymethyl cellulose (CMC, Alpha Aesar, lot no.: 10193024, expiry date 03/2021) added to the polar extract to a final concentration of 2%
Concentration:
0, 12.5, 25 and 50% (w/v)
No. of animals per dose:
5 mice / group
5 mice / prescreen test
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in Aqua ad injectionem (AlleMan Pharma, lot no. 601101, expiry date: 10/10/2016) containing 2% CMC
- Irritation:
4 animals were treated by topical application with the test item (25 µL of the suspension, 2 mice per dose (50% (w/v), 25% (w/v))) on three consecutive days to the entire dorsal surface of each ear. One further animal was treated with the vehicle Aqua ad injectionem and served as negative control.
- Systemic toxicity:
During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
- Ear thickness measurements: day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6 (shortly before sacrificing)
- Erythema scores: see Table 1 in box "Any other information in material and methods"
Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%

For the results of the pre-screen tests please refer to the box "Any other information on results"

MAIN STUDY

- Controls
Aqua ad injectionem containing 2% CMC was used as vehicle and served as negative control. Positive controls with α-Hexyl Cinnamaldehyde in Aqua ad injectionem containing 2% CMC are performed periodically. The recent results are included in the report
-Other Materials
3H-methyl thymidine (TRK 300, 20 Ci/mmol; PerkinElmer, lot no. 201604E), diluted to a working concentration of 80 µCi/mL.
Phosphate buffered saline (PBS), Eurofins Munich, lot no. 30082016, expiry date: 26/03/2017.
Trichloroacetic acid (TCA), Sigma-Aldrich, lot no. BCBN7603V, expiry date: 15/05/2017.
- Preparation of the Test Item
The preparations were made immediately prior to each dosing.
- Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.). Identification was ensured by cage number and individual marking (tail).
- Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.
- Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).
- Test Regime
-> Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.
-> Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.
-> Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
-> Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
- Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3= c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, sixth revised edition, 2015:
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The positive-control substance exceeded the stimulation index of 3 (see Table 2 in box "Any other information on results")
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
0.9
Variability:
SD = 0.2
Test group / Remarks:
12.5% (w/v)
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
0.8
Variability:
SD = 0.2
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
1.2
Variability:
SD = 0.3
Test group / Remarks:
50% (w/v)
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Please see Table 3 in box "Any other information on results"

EC3 CALCULATION
The EC3 value could not be calculated as the stimulation idices of all concentrations were below 3.

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs except of the animals treated with the test item at the concentration of 50% (w/v), which showed erythema grade 1 at the application sites from day 3 until day 5 and alopecia on days 3, 4, and 6.

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Results of the pre-screen tests

Solubility tests

The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in aqua ad injectionem containing 2% CMC

Irritation and toxicity test

Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. All animals showed the expected weight development.

Results of the main study

Table 2: Stimulation Indices obtained for the Positive-Control Group

Test Item Concentration
[%]
Animal Number Stimulation Index
Negative Control Aqua ad injectionem with CMC (2%) final 100 601
602
603
604
605
MV 1.0
SD
Positive Control
  1. α-Hexyl Cinnamic
    aldehyde
    in Aqua ad injectionem with CMC (2%) final
100 606 7.7
607 7.8
608 11.4
609 10.3
610 12.7
MV 10.0
SD 2.0

SD = standard deviation; MV = mean value

Table 3: Stimulation Indices obtained in the main experiment

Test Item Concentration
[%]
Animal Number Stimulation Index
Negative Control Aqua ad injectionem with CMC (2%) final 100 601
602
603
604
605
MV 1.0
SD
1-Benzyl-3-Carbamoyl-pyridinium, chloride in Aqua ad injectionem with CMC (2%) final 12.5 1 0.5
2 0.9
3 1.1
4 0.7
5 1.1
MV 0.9
SD 0.2
1-Benzyl-3-Carbamoyl-pyridinium, chloride in Aqua ad injectionem with CMC (2%) final 25 6 0.8
7 0,6
8 1
9 n.d.*
10 0.6
MV 0.8
SD 0.2
1-Benzyl-3-Carbamoyl-pyridinium, chloride in Aqua ad injectionem with CMC (2%) final 50 11 1.6
12 1.2
13 1.3
14 1.4
15 0.8
MV 1.2
SD 0.3

* = outlier, failed Grubbs, Nalimov and Dixon; n.d. = not determined; SD = standard deviation; MV = mean value

If not noted individually, the results include both lymph nodes of an animal.

Interpretation of results:
GHS criteria not met
Conclusions:
1-Benzyl-3-carbamoyl-pyridinium, chloride is not sensitizing under the conditions of this LLNA study (OECD 429).
Executive summary:

In a dermal sensitization study conducted according to OECD 429 with 1-Benzyl-3-Carbamoyl-pyridinium, chloride (approx. 96 % purity) suspended in Aqua ad inject. containing CMC (2% final), young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 12.5% (w/v), 25 % (w/v) and 50 % (w/v) in a local lymph node assay (LLNA). Due to animal welfare reasons the negative control was shared and a periodically performed positive control (α-Hexyl Cinnamicaldehyde in Aqua ad injectionem with CMC (2%) final) was used. There was no mortality and no significant clinical observations or effects on body weights except of the animals of the high dose group showing erythema grade 1 at the application side from day 3 until day 5 and alopecia on days 3, 4 and 6. None of the tested concentrations of the test substance reached the stimulation index threshold of 3. In this study, 1-Benzyl-3-Carbamoyl-pyridinium, chloride is not a dermal sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a dermal sensitization study according to OECD 429 1-Benzyl-3-carbamoyl-pyridinium, chloride (target substance) was tested at concentrations of 12.5% (w/v), 25 % (w/v) and 50 % (w/v). There was no mortality and no significant adverse clinical signs of toxicity or adverse effects on body weights were observed. None of the tested concentrations of the test substance reached the stimulation index threshold of 3. Based on the results 1 -Benzyl-3 -carbamoyl-pyridinium, chloride can be considered as not dermal sensitizing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a dermal sensitization study according to OECD 429, female mice were tested negative for 1-Benzyl-3-carbamoyl-pyridinium, chloride. Based on the results, no classification of the test item is warranted.