Rosin, decarboxylated

Endpoint summary

Description of key information

OECD 429 (LLNA): Skin Sensitiser 1 (H317: May cause an allergic skin reaction)

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:

2020-09-24 to 2021-02-11

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

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, Blackthorn
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Only healthy animals were used for the study.
- Age at study initiation: 9-10 weeks old on Day 1
- Weight at study initiation: 19 to 23g on Day 1

- Housing: Group caging (up to five during acclimatisation) in cages that conformed to the ‘Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014). From Day 1, the preliminary study animal was individually housed and the main study animals were housed in groups of up to three.
- Diet (e.g. ad libitum): 5LF2 EU Rodent Diet 14% (provided by LabDiet, PMI Nutrition), ad libitumm.
- Water (e.g. ad libitum): tap water from the municipal supply from 500 mL bottles, ad libitum.
- Acclimation period:8 to 16 days
- Indication of any skin lesions: Not specified
but only healthy animals were used for the study. Health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

- IN-LIFE DATES: 2020-10-07 To: 2020-10-21

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Test material formulations at 100%, 50% and 25% in AOO (acetone/olive oil (4:1 v/v))

No. of animals per dose:

5/dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:
The solubility of the test material was determined in acetone/olive oil 4:1 v/v. This vehicle was chosen as this is one of the standard OECD vehicles and the formulation produced an overtly stable solution, emulsion or dispersion incorporating
50% v/v of the test article.

The test material formulations were prepared daily

- Irritation:
In the Preliminary Irritation / Toxicity Test, a single mouse was treated by daily application of 25 μL of the undiluted test article to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and scored.
- Ear thickness measurements: Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.

- Erythema scores: Both ears of the mouse were observed for erythema and scored using the criteria detailed below:
Erythema Scoring
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 4
Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Three consecutive concentrations were selected on the basis of the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation. Mice were allocated into groups of five female mice. 100, 50 and 25% of the test formulations were selected as the most appropriate concentrations for the main study. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate test or control formulation (25 μL/pinna) dispensed from an automatic micro pipette.

TREATMENT PREPARATION AND ADMINISTRATION:
The five groups of five female mice were subjected to application of the vehicle control, positive control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3. On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection.

Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exsanguination under a deep plane of inhalation anaesthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

CLINICAL SIGNS: Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article.

BODY WEIGHTS: Mice were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of 3HTdR.



On the following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial containing ca 10mL scintillation fluid and counted by Liquid scintillation (LSC).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Number of radioactive disintegrations per minute (DPM) were measured for each individual animal. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value.

Stimulation index (SI = DPM value of each treated group divided by the mean DPM value of the negative control group) for each treatment group was also calculated.

As a Stimulation Index value of 3 or more was obtained in all groups, DPM values were analysed using the methods detailed below.

Tests were performed using a one-sided risk for increased response with increasing dose.

The positive control group was excluded from statistical analyses. The vehicle control group was taken as the baseline group with which the treated groups were compared.

Significant results are reported as P<0.05, P<0.01 or P<0.001.

Positive control results:

The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0. The positive control article produced a Stimulation Index of 8.81, demonstrating adequate performance of the assay.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control (AOO)

Key result

Parameter:

SI

Value:

6.54

Test group / Remarks:

25% v/v

Key result

Parameter:

SI

Value:

13.69

Test group / Remarks:

50% v/v

Key result

Parameter:

SI

Value:

14.84

Test group / Remarks:

100% v/v

Key result

Parameter:

SI

Value:

8.81

Test group / Remarks:

Positive control

 

Table 1 – Preliminary Screening Test – Observations, Body Weights and Mortality Data

Concentration %	Animal Number	Body Weight (g) on Day:		Observations (Days)
		1	6	1	2	3	4	5	6
100	583	22	21	GF	GF	GF	GF	GF, DS	GF, DS

Key

DS – Dry skin behind ears

GF – Greasy fur behind ears, top of head and back of neck

 

Table 2 – Preliminary Screening Test – Erythema

Concentration %	Animal Number	Grade of Erythema on Day:
		1	2	3	4	5	6
100	583	0	0	2	1	1	0

Key

0 – No erythema

1 – Very slight erythema (barely perceptible)

2 – Well- defined erythema

 

Table 3 – Preliminary Screening Test – Erythema

Concentration %	Animal Number	Ear	Thickness (mm) on Day:
			1	3	6
100	583	Left	0.24	0.24	0.23
		Right	0.21	0.21	0.23

 

Table 4 – individual DPMs and Stimulation Index (SI)

 

Concentration (% v/v in AOO 4:1 (v/v))	Group Number	Animal Number	DPM / Animal	Mean DPM / Animal (Standard Deviation)	Stimulation Index (SI) ^a
Vehicle	1	584 585 586 587 588	317 407 384 332 267	341 (±55.5)	NA
25	2	589 590 591 592 593	2064 3003 3376 2124 602	2234 (±1072.4) *	6.54
50	3	594 595 596 597 598	2876 4714 5703 4649 5422	4673 (±1101.5) ***	13.69
100	4	599 600 601 602 603	4993 5402 2932 4739 7260	5065 (±1548.6) ***	14.84
Positive control	5	604 605 606 607 608	2792 1627 2416 3902 4306	3009 (±1094.1)	8.81

^a = Stimulation Index of 3.0 or greater indicates a positive result

AOO = Acetone in olive oil 4:1 (v/v)

NA = Not applicable

* = Significantly different from control group p<0.05

*** = Significantly different from control group p<0.001

 

 

Table 5 – Body weights

Concentration (% v/v in AOO 4:1 (v/v))	Group Number	Animal Number	Body Weights (g)
			Day 1	Day 6
Vehicle	1	584 585 586 587 588	21 21 21 20 22	21 23 21 22 24
25	2	589 590 591 592 593	22 19 22 20 23	23 21 24 21 23
50	3	594 595 596 597 598	23 23 20 21 22	22 24 21 20 23
100	4	599 600 601 602 603	22 21 21 20 21	22 22 22 21 22
Positive control	5	604 605 606 607 608	22 23 20 22 20	22 22 22 22 22

 

AOO – Acetone in olive oil 4:1 (v/v)

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the conditions of the Local Lymph Node Assay, it was demonstrated that AA-948-61 (Decarboxylated Rosin, CAS No. 8050-18-8) has the potential to cause skin sensitisation.

Executive summary:

A key OECD 429 study was conducted to assess the potential of the test article, AA-948-61 (Decarboxylated Rosin, CAS No. 8050-18-8), to cause skin sensitisation in the mouse.

 

Following a preliminary screening test using the undiluted test article, the main study was conducted using the undiluted test article and 50 and 25% v/v formulations in acetone/olive oil 4:1 (v/v).

Groups of five female CBA/CaOlahsd mice were subjected to topical applications of positive control, vehicle control or of one the test formulations (100, 50 or 25%) to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3.

 

On Day 6, a 20 μCi dose of tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter for each animal.

 

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

The stimulation index values were 14.84, 13.69 and 6.54 at concentrations of 100%, 50% (w/v) and 25% (w/v), respectively.

Under the conditions of the Local Lymph Node Assay, it was demonstrated that AA-948-61 (Decarboxylated Rosin, CAS No. 8050-18-8) has the potential to cause skin sensitisation.

 

The positive control substance produced a Stimulation Index of 8.81, demonstrating adequate performance of the assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A key OECD 429 study was conducted to assess the potential of the test article, AA-948-61 (Decarboxylated Rosin, CAS No. 8050-18-8), to cause skin sensitisation in the mouse.

 

Following a preliminary screening test using the undiluted test article, the main study was conducted using the undiluted test article and 50 and 25% v/v formulations in acetone/olive oil 4:1 (v/v).

Groups of five female CBA/CaOlahsd mice were subjected to topical applications of positive control, vehicle control or of one the test formulations (100, 50 or 25%) to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3.

 

On Day 6, a 20 μCi dose of tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter for each animal.

 

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

The stimulation index values were 14.84, 13.69 and 6.54 at concentrations of 100%, 50% (w/v) and 25% (w/v), respectively.

Under the conditions of the Local Lymph Node Assay, it was demonstrated that AA-948-61 (Decarboxylated Rosin, CAS No. 8050-18-8) has the potential to cause skin sensitisation. Based on the results observed, AA-948 -61 meets the criteria to be classified as a skin sensitiser Category 1 (H317: May cause an allergic skin reaction) under EU REgulation (EC) No 1272/2008 (CLP) and GHS (rev.7) 2017.

 

The positive control substance produced a Stimulation Index of 8.81, demonstrating adequate performance of the assay.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

AA-948 -61 meets the criteria to be classified as a skin sensitiser Category 1 (H317: May cause an allergic skin reaction) under EU REgulation (EC) No 1272/2008 (CLP) and GHS (rev.7) 2017.