Rosin, decarboxylated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-27 to 2020-10-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Decarboxylated Rosin

Cas Number:

8050-18-8

Molecular formula:

Not availabe for UVCB substance

IUPAC Name:

Decarboxylated Rosin

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir (Woolley Brothers, Sheffield) as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of test item

Duration of treatment / exposure:

exposure period of 10 minutes

Details on study design:

1 – Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


2 – selection of corneas and opacity reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (Annex 2).
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.


3 – Treatment of cornea
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.

At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.


4 – Application of sodium fluroscein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.


5 – Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.


6 – Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. With a clear negative result obtained it was considered that histopathology would not be required.


7 – Data Evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.


8 – Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.


9 – Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.


10 – In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.


11 – Visual Observation
The condition of the cornea was visually assessed post treatment and post incubation.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control

Value:

41.4

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative control

Value:

0.1

Negative controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

2

Other effects / acceptance of results:

For an acceptable test the following positive control criterion should be achieved:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.

Ethanol (a known ocular irritant was included to verify that an appropriate response was induced. If the BCOP assay is being used only to identify corrosive or severe irritants, then the positive control substance should induce a severe response in vivo as well as in the BCOP assay. However, to ensure variability in the positive control response across time can be assessed, the magnitude of the severe response should not be excessive.


For an acceptable test the following negative control criterion should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values calculated from the previous 12 months data for this testing facility.

Any other information on results incl. tables

Table 1 – individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In Vitro Irritancy Score
		Pre -Treatment	Post - Treatment	Post Incubation	Post Incubation – Pre- Treatment	Corrected Value		Corrected Value
Negative Control	2	4	3	3	0		0.012
	3	4	3	3	0		0.006
	6	3	3	3	0		0.011
					0.0 *		0.010 ◊		0.1
					0		0.003 ▪
Positive Control	9	5	34	30	25	25.0	0.755	0.745
	11	4	36	34	30	30.0	1.157	1.147
	12	3	31	29	26	26.0	0.999	0.989
						27.0 ●		0.961 ●	41.4
						2.6 ▪		0.203 ▪
Test Item	14	3	5	5	2	2.0	0.014	0.004
	15	4	5	5	1	1.0	0.014	0.004
	16	4	6	7	3	3.0	0.009	0.000
						2.0 ●		0.003	2.0
						1.0 ▪		0.0 ▪

 

OD = Optical density

* = Mean of the post-incubation – pre-treatment values

◊ = Mean permeability

● = Mean corrected value

▪ = Standard deviation

 

Table 2 – Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment	Cornea Number	Observations
		Post Treatment	Post Incubation
Negative Control	2	Clear	Clear
	3	Clear	Clear
	6	Clear	Clear
Positive Control	9	Cloudy	Cloudy
	11	Cloudy	Cloudy
	12	Cloudy	Cloudy
Test Item	14	Clear	Clear
	15	Clear	Clear
	16	Clear	Clear

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to UN GHS Classification, the test item AA-948-61 (Decarboxylated Rosin, CAS no. 8050-18-8) is given No Category classification under the conditions of the test.

Executive summary:

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The in vitro irritancy scores are summarised as follows: test item – 2.0; negative control – 0.1; positive control – 41.4.

According to UN GHS Classification, the test item AA-948-61 (Decarboxylated Rosin, CAS no. 8050-18-8) is given No Category classification under the conditions of the test.