Pyrolytic sugar

Endpoint summary

Description of key information

Two in vitro studies, Lu-Sens and U-Sens were ran to assess the skin sensitisation potency of the test substance in a weight of evidence approach. The 2 studies conducted represent 2 key events in the adverse outcome pathway: Lu-Sens representing the 2nd key event (Keratinocyte induction of cyto-protective gene pathways) and U-sens the 3rd key event (activation of dendritic cells). Both studies give indication that the test item might be skin sensitizing, therefore it was concluded to regard the substance as skin sensitizing category 1.

 

Subsequently, an in vivo LLNA study was performed in accordance with OECD 429. This study confirmed the skin sensitizing potential of the substance. The study yielded stimulation indices (SI) of 9.10 at a concentration of 5% w/v, 20.72 at 10% w/v and 20.81 at 25% w/v. The EC3 value was determined to be 2.38%, which allocates the test item to skin sensitizing category 1B.

 

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:

January 2022

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)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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: 8 weeks (Pre-screen test), 9 weeks (Main study);
- Housing: The animals were housed in TECNIPLAST cages from the Tecniplast Company, in Conventional animal room No. B2-611. 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

Vehicle:

dimethyl sulphoxide

Concentration:

Group 1: negative control.
Group 2: positive control.
Group 3: test item 5% w/v.
Group 4: test item 10% w/v.
Group 5: test item 25% 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: 6 animals per dose
main study: 5 animals per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
- 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.
- Each day between 9-10 a.m., 25 µL of the test substance (as a 100%, 50% or 25% w/v solution) or control solutions 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
- 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.

TREATMENT PREPARATION AND ADMINISTRATION:
- Each day, 0.2g of the test item was dissolved in 0.8 mL of DMSO vehicle (25% w/v stock solution) one hour before the application to an ear of the mice. 10% and 5% solutions were prepared by dilution of stock solution.
- 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.

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.

Positive control results:

Stimulation index SI = 9.86 for positive control (alpha-Hexylcinnamic aldehyde) was in the middle range of historical positive controls

Parameter:

SI

Value:

20.81

Test group / Remarks:

25%

Parameter:

SI

Value:

9.1

Test group / Remarks:

5%

Parameter:

SI

Value:

20.72

Test group / Remarks:

10%

Key result

Parameter:

EC3

Value:

2.38

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The pooled lymph node weights of treated groups were 0.0491 g for 5 % (w/v) concentration, 0.0580 g for 10 % (w/v) concentration and 0.0738 g for 25 % (w/v) concentration of the test item. The lymph node weights of negative control group and positive control group were 0.0287 g and 0.0485 g, respectively. The DPM values for the three treated groups were 13 247 (5 % w/v), 30 171 (10 % w/v) and 30 303 (25 % w/v), respectively. The SI values for the three treated groups were 9.10 (5 % w/v), 20.72 (10 % w/v) and 20.81 (25 % w/v), respectively. The EC3 value was calculated to be 2.38 %.

CLINICAL OBSERVATIONS: No signs of significant local irritation except slight vascular drawing (1 animal, day 3) and small scabs on the ears (1 animal, days 5-6) were observed in group treated with low dose of BTG Pyrolytic Sugar (5 % w/v). In middle-dosed animals (PS 10 % w/v), vascular drawing (all 5 animals) rather than erythema (1 animal only) was visible on ears on days 3-4. Erythema (score 2, well defined) accompanied with scales (1 animal) and scabs (1 animal) and significant vascular drawing were recorded in high dose (25 % w/v) treated animals on days 3-6.
Signs of local irritation were observed in positive control group. Moderate redness (score 2-3) accompanied with vascular drawing was observed on days 2-6. From day 5, ear skin was covered with scales (5 animals) or scabs (1 animal).

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 Sugar (PS 5 % w/v and 10 % w/v). In high-dosed group (PS 25 % w/v), slight behavioural changes, irritation and touch sensitivity were observed on days 3,4.
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), grooming activity and touch sensitivity 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 affect 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.

Pre-screen test

To determine the highest tolerated and non-irritant test concentration a Pre-screen test was performed. Pyrolytic Sugar was dissolved in DMSO and administered at concentrations of 100 %, 50 % and 25 % w/v. The daily clinical observation of the animals was performed to monitor clinical signs of toxicity. In high dosed group (PS 100 % w/v), changes in behaviour occurred and separation of animals from each other was observed. Slight behavioural changes - irritation and touch sensitivity were found also in animals dosed with 50% and 25% w/v. 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. On days 3, 5-6, slight erythema (score 1) was observed in mice after administration of low dose of the test item (PS 25 % w/v). Well defined erythema (score 2) was visible in this group only on day 4. In middle-dosed animals (PS 50 % w/v), erythema score 2 (well defined erythema) was visible on the ears of mice on days 2 and 4.  Erythema worsened to score 3 (moderate erythema) on day 3. Erythema was accompanied with slight vascular drawing, mainly on days 4-5. In high-dosed animals, starting from day 2, erythema score 2 was observed (PS 100 % w/v). On day 4-6, the erythema score worsened to 3 or 3-4 (moderate to severe erythema) with scab formation. From day 3, significant vascular drawing on the ears was also observed.

On day 1 (pre dose), day 3 and day 6, the ear thickness was measured. The maximum increase in ear thickness was 20 % recorded in low dosed group of mice (PS 25 % w/v) on day 6.

In conclusion, erythema reached score 3 and 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	0.0287	1 456		-
Positive control	0.0485	14 355	9.86	-
5 % (w/v) Pyrolytic Sugar	0.0491	13 247	9.10	2.375
10 % (w/v) Pyrolytic Sugar	0.0580	30 171	20.72	-
25 % (w/v) Pyrolytic Sugar	0.0738	30 303	20.81	-

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

 

 

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The skin sensitizing potential of Pyrolytic Sugar was assessed using the murine local lymph node assay. Based on the results of this study, Pyrolytic Sugar is considered a skin sensitizer under the conditions of this LLNA study. According to the ECETOC classification, test substance Pyrolytic Sugar is classified as moderate allergen.

Executive summary:

The sensitization potential of Pyrolytic Sugar (PS) 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 (alpha-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 % and 25 % (w/v). Based on the observation of changes in behaviour (systemic toxicity) and of erythema score 3 in middle dosed and high dosed group of mice (PS 50 % w/v, PS 100 % w/v) in the Pre-screen test, the concentrations of 5 %, 10 % and 25 % (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 5 %, 10 % and 25 % (w/v) in DMSO. Positive control was exposed to the 25 % solution of alpha-hexylcinnamic aldehyde in DMSO and 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.

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 Sugar (PS 5 % w/v and 10 % w/v). In high-dosed group (PS 25 % w/v), slight behavioural changes, irritation and touch sensitivity were observed on days 3,4.

No signs of significant local irritation were observed in group treated with low dose of BTG Pyrolytic Sugar (5 % w/v). In middle-dosed animals (PS 10 % w/v), vascular drawing rather than erythema was visible on ears on days 3-4. Significant vascular drawing and erythema score 2, randomly accompanied with scales and scabs were recorded on days 3-6 in high dose (25 % w/v) treated animals.

In this study, the Stimulation Indices (SI) of 9.10, 20.72 and 20.81 were determined with the test item at concentrations of 5%, 10%, and 25% (w/v) BTG Pyrolytic Sugar in DMSO, respectively. The EC3 value was calculated to be 2.38 %.

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

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

Based on the EC3 value of 2.38% as determined in the in vivo LLNA study, the test item is to be allocated to skin sensitizing category 1B.