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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:
January 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Low molecular weight organic substances from fast pyrolysis bio-oil
IUPAC Name:
Low molecular weight organic substances from fast pyrolysis bio-oil
Constituent 2
Reference substance name:
Monomers from fast pyrolysis bio-oil
IUPAC Name:
Monomers from fast pyrolysis bio-oil
Constituent 3
Reference substance name:
Dimers from fast pyrolysis bio-oil
IUPAC Name:
Dimers from fast pyrolysis bio-oil
Constituent 4
Reference substance name:
Trimers from fast pyrolysis bio-oil
IUPAC Name:
Trimers from fast pyrolysis bio-oil
Constituent 5
Reference substance name:
Higher oligomers from fast pyrolysis bio-oil
IUPAC Name:
Higher oligomers from fast pyrolysis bio-oil
Constituent 6
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
Test material form:
liquid: viscous
Remarks:
Brown /black highly vicous liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Anlab, Praha, Czech Republic
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Animals were healthy, without visible symptoms of disease.
- Age at study initiation: 9 weeks (Pre-screen test), 10 weeks (Main study);
- Housing: The animals were housed in TECNIPLAST cages from the Tecniplast Company, in Conventional animal room No. B2-610. Maximum six females were housed per cage. The room temperature was within the range of 20-24°C, relative humidity was within the range of 40-60 %. The animals were subjected to a 12-hour light/ 12-hour dark cycle. The sanitation was performed according to SOP ŠPP/EZ/V005.
- Diet: A laboratory food for mice V1534-000 R/M-H (Ssniff) was served ad libitum. The certificate of analysis is included in the raw data.
- Water: The animals received tap water for human consumption ad libitum. The water from the local mains was monitored for quality by testing for the microbiological and chemical quality by Waterworks Bratislava quarterly. Bottles were exchanged and cleaned once a week. The quality of drinking water is periodical monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
- Acclimation period: The animals were acclimated to the conditions identical to the conditions during the experiment five days prior to the start of treatment. The acclimation was performed according to standard operation procedure ŠPP/EZ V001
- Indication of any skin lesions: the health condition of animals and skin integrity was examined by a veterinarian before initiation of the study (Pre-screen test and Main study).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-60 %
- Photoperiod (hrs dark / hrs light): 12-hour light/ 12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Pre-screening study 1: 100% w/v, 50% w/v and 25% w/v
Pre-screening study 2: 10% w/v and 5% w/v
Main study: 10% w/v, 5% w/v and 2.5% w/v

The doses were selected from the concentration series 100 %, 50 %, 25 %, 10 %, 5 %, 2.5 % etc. according to OECD Guideline No. 429. The starting concentration of test item in the Main Study was determined according to Pre-screen test results.
No. of animals per dose:
pre-screen test: 10 animals per dose
main study: 5 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: H2O: unknown; EtOH: > 1 g/L; acetone: > 1 g/L; CH3CN: unknown; DMSO: > 1 g/L
- For Pre-screen 1: Each day, 0.5g of test item was dissolved in 0.5 mL of DMSO vehicle (100% w/v stock solution) one hour before the application to an ear of the mice. 50% and 25% solutions were prepared by dilution of stock solution.
- For Pre-screen 2: Each day, 0.05g of test item was dissolved in 0.5 mL of DMSO vehicle (10% w/v stock solution) one hour before the application to an ear of the mice. 5% solution was prepared by dilution of stock solution.
- Each day between 9-10 a.m., 25 µL of the test substance (as a 100%, 50%, 25%, 10% or 5% w/v solution) was administered to the dorsum of each ear.
- 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 on day 1 before application and prior to the termination (day 6). Both ears of each mouse were observed for erythema and scored. Ear thickness was measured by a digital micrometer on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 % in day of measurement.

MAIN STUDY
Each day, 0.1g of test item was dissolved in 1 mL of DMSO vehicle to yield the 10% w/v solution. For the 5% w/v solution, 0.05g of test item was dissolved in 1 mL of DMSO. For the 2.5% w/v solution, 0.025g of test item was dissolved in 1 mL of DMSO. Solutions were prepared one hour before the application to an ear of the mice.

Each day between 9-10 a.m., the test substance in three different concentrations was administrated in volume of 25 µL each to the dorsum of each ear. The alpha-Hexylcinnamaldehyde (25 %) in DMSO as positive control, and vehicle (DMSO) as negative control were administrated in the same volume.

Day 1: Each animal was identified, and the body weight was recorded. To the dorsum of each ear 25 µL of the appropriate dilution of the test item, positive control, 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. 250 μL of sterile phosphate-buffered saline (PBS) containing 20 μCi (7.4×105 Bq) of tritiated (3H)-methyl thymidine was injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).


ANIMAL ASSIGNMENT AND TREATMENT
Before the treatment animals were randomized based on body weights to treatment groups.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the pooled DPM of the vehicle control group; this yields a mean SI. A test item is regarded as sensitizer in the LLNA test when SI ≥ 3.
Estimated concentration three (EC3): Estimated concentration of a test substance needed to produce a stimulation index of three. EC3 value 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.

Results and discussion

Positive control results:
Stimulation index SI = 4.63 for positive control (alpha-Hexylcinnamic aldehyde) was in the middle range of historical positive controls

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
3.02
Test group / Remarks:
10%
Parameter:
SI
Value:
1.74
Test group / Remarks:
5%
Parameter:
SI
Value:
1.34
Test group / Remarks:
2.5%
Key result
Parameter:
EC3
Value:
10
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: The pooled lymph node weights of treated groups were 0.0332 g for 2.5 % (w/v) concentration, 0.0374 g for 5 % (w/v) concentration and 0.0483 g for 10 % (w/v) concentration of the test item. The lymph node weights of negative control group and positive control group were 0.0336 g and 0.0637 g, respectively. The DPM values for the three treated groups were 16 911 (2.5 % w/v), 21 952 (5 % w/v) and 38 006 (10 % w/v), respectively. The SI values for the three treated groups were 1.34 (2.5 % w/v), 1.74 (5 % w/v) and 3.02 (10 % w/v), respectively. The EC3 value is equal to 10 %.

CLINICAL OBSERVATIONS: No signs of significant local irritation except slight vascular drawing (day 2-4) was observed in group treated with middle dose of BTG Pyrolytic Lignin (5 % w/v). In high-dosed animals (10 % w/v), erythema score 2 (well-defined) and vascular drawing were visible on ears on days 2-6.
Signs of local irritation were observed in positive control group. Moderate redness (score 2-3) accompanied with vascular drawing was observed on days 2-5.

SIGNS OF TOXICITY : Daily clinical observation of animals did not show visible clinical signs of systemic toxicity in mice administered with low and middle dose of BTG Pyrolytic Lignin (2.5 and 5 % w/v). In animals dosed with 10 % w/v, slight behavioural changes displayed as irritation on days 2,3.
Signs of systemic toxicity were observed in the positive control group. On days 2-4, changes in posture (hunched posture), social behaviour (separation from group), and grooming activity were observed in positive control group.

BODY WEIGHTS: The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. Administration of the test item in used concentrations did not affected the body weight of treated animals.
No differences were found in body weight gain between initial body weight (day 1) and terminal body weight (day 6). Similarly, no significant changes in body weight between treated groups and negative (vehicle) control at day 1 and day 6 of the study were recorded.

Any other information on results incl. tables

Pre-screen test


To determine the highest tolerated and non-irritant test concentration a Pre-screen test was performed. Pyrolytic Lignin was dissolved in DMSO and administered at concentrations of 100 %, 50 %, 25 %, 10 % and 5 % w/v. The daily clinical observation of the animals was performed to monitor clinical signs of toxicity. In the highest dosed group (100 % w/v) as well as in 50 % and 25% groups, changes in behaviour occurred and separation of animals from each other, hunched position, blepharospasm and sensitivity of response to touch of the ears were observed. At the lower dose (10%), sensitivity still persisted. The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes in body weights were observed during the Pre-screen test.


Both ears of each mouse were observed for erythema and scored. In the highest dosed group (100 % w/v) score reached 3-4 (moderate to severe erythema with maximum on days 4-6. From day 3, ear skin was covered with scabs (1 animal) and small alopetic spot on the head of the animal was observed. In group administered with 50 %, moderate to severe erythema (score 3) was found (days 4-6). Erythema in lower groups (25% and 10%) reached score 2 (well defined erythema). Erythema was accompanied by significant vascular drawing of similar dose-dependent intensity as concomitant erythema.


On day 1 (pre dose), day 3 and day 6, the ear thickness was measured.


The maximum increase in ear thickness was 42% recorded in high dosed group of mice (100 % w/v) on day 3.


In conclusion, the erythema reached score 3 in groups 100% and 50%. Ear thickness reached 42% increase in group 100%, 28% enhancement in group 50% and 41% increase in group 25%. These groups met the criteria which are considered as signs for excessive local skin irritation (erythema score ≥ 3 and/or ear thickness increase ≥ 25 %).


MAIN STUDY


Lymph node weight, Proliferative activity (DPM), Stimulation Index (SI), EC3 value
















































Group



Lymph node weight (g)



DPM



SI



EC3 (%)



Negative control



0.0336



12 591



 



 



Positive control



0.0637



58 310



4.63



 



2.5 % (w/v) Pyrolytic Lignin



0.0332



16 911



1.34



 



5 % (w/v) Pyrolytic Lignin



0.0374



21 952



1.74



 



10 % (w/v) Pyrolytic Lignin



0.0483



38 006



3.02



10



Legend: DPM - disintegrations per minute, EC3 - estimated concentration three


 


 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The skin sensitizing potential of Pyrolytic Lignin was assessed using the murine local lymph node assay. Based on the results of this study, Pyrolytic Lignin is considered a skin sensitizer under the conditions of this LLNA study. According to the ECETOC classification, test substance BTG Pyrolytic Lignin is classified as weak allergen.
Executive summary:

The sensitization potential of Pyrolytic Lignin (PL) was evaluated using Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals.


Based on the recommendations of the OECD Guideline No. 429, the test item was dissolved in dimethyl sulphoxide (DMSO). The positive control (a-Hexylcinnamic aldehyde) was dissolved in DMSO. Selection of the doses was limited by suspensibility of the test item in recommended vehicles. The Pre-screen test was performed using the concentrations of 100 %, 50 %, 25 %, 10 % and 5 % (w/v). In Pre-screen test, erythema score 3 in groups of mice dosed with 100 %, 50% and 25% w/v was found. Moreover, mean increase in ear thickness 29 %, 28 % and 41 % in groups of mice dosed with 100 %, 50% and 25% w/v, respectively was observed. Based on these observations in the Pre-screen test, the concentrations of 10 %, 5 % and 2.5 % (w/v) were selected for the Main study.


Five female mice (CBA/CaOlaHsd) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 10 %, 5 % and 2.5 % (w/v) in DMSO. Positive control was exposed to the 25 % solution of a-Hexylcinnamic aldehyde in DMSOand negative control to the vehicle (DMSO), only. Lymphocyte proliferation was measured using incorporation of radioactive 3H-methyl-thymidine in the draining lymph nodes. The radioactive incorporation was measured as disintegrations per minute (DPM). Results were expressed as Stimulation Index (SI) calculated as DPM/pooled treatment group divided by DPM/vehicle control group.


Regardless of the dose applied, daily clinical observation of animals did not show visible clinical signs of systemic toxicity in all Pyrolytic Lignin treated mice. No signs of significant local irritation were observed in test item treated groups. Slight vascular drawing was visible on ears of mice in high dosed group (10 % w/v) on days 2-3. 


In this study, the Stimulation Indices (SI) of 1.34, 1.74 and 3.02 were determined with the test item at concentrations of 2.5 %, 5 %, and 10 % (w/v) Pyrolytic Lignin in DMSO, respectively. The EC3 value was calculated to be 10 %.


The test item Pyrolytic Lignin is considered a skin sensitizer under the test conditions of this study. According to the ECETOC classification, test substance Pyrolytic Lignin is classified as weak allergen.