Rosin, decarboxylated

Endpoint summary

Description of key information

In a key OECD 431 and 439 study, Decarboxylated Rosin (CAS No. 8050 -18 -8) was demonstrated not corrosive and not irritating to the skin. In addition, a key OECD 437 BCOP eye irritation study, Decarboxylated Rosin (CAS No. 8050 -18 -8) was considered to be not irritating to the eye (UN GHS Classification: No Category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-06 to 2020-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

0.9 mL of this assay medium at room temperature, was pipetted into the wells of two pre-labeled 6-well plates.

Duration of treatment / exposure:

The tissues were exposed to the test item for a 3-Minute and 60-Minute period.

Duration of post-treatment incubation (if applicable):

The rinsed tissues were incubated at 37 C°, 5% CO2 in air for 3 hours.

Number of replicates:

In duplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minutes

Value:

98.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 minutes

Value:

93.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD570 for the negative control treated tissues was 1.884 for the 3-Minute exposure period and 2.067 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.0% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Table 1 - Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	Mean OD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.979	1.884	0.135	7.2	100*
		1.788
	60 Minutes	2.040	2.067	0.038	1.8
		2.094
Positive Control	3 Minutes	0.071	0.065	0.008	NA	3.5
		0.059
	60 Minutes	0.078	0.063	0.021	NA	3.0
		0.048
Test Item	3 Minutes	2.017	1.848	0.239	12.9	98.1
		1.679
	60 Minutes	2.100	1.931	0.240	12.4	93.4
		1.761

*=          The mean viability of the negative control tissues is set at 100%

Conclusions:

In this study and under the experimental conditions reported:
The test item was considered to be non-corrosive to the skin.
EU CLP Not classified for corrosion.

Executive summary:

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able to reduce MTT to formazan whereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Duplicate tissues were treated with the negative control, positive control and test item for exposure periods of 3 and 60 minutes. As the test item had been shown to directly reduce MTT additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viabilities for the test item treatment groups were 98.1 and 93.4% for 3 and 60 minute exposures respectively. The mean viability of the negative control tissues is set at 100% and positive control tissues elicited a positive response with percentage viabilities of 3.5 and 3.0% for 3 and 60 minute exposures respectively.The criteria required for acceptance of results in the test were satisfied.

The results obtained showed that no negligible interference due to direct reduction of MTT by the test item occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.

In conclusion, this study and under the experimental conditions reported the test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-14 to 2020-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes

Duration of post-treatment incubation (if applicable):

The rinsed tissues were incubated at 37 C°, 5% CO2 in air for 42 hours.

Irritation / corrosion parameter:

other: Optical Density at 570 nm

Value:

0.743

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 8.7 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.2 %. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.729 and the standard deviation value of the viability was 5.9 %. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.1 %. The test item acceptance criterion was therefore satisfied.

Table 1 - Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.732	0.729	0.043	100.4	100^*	5.9
	0.684			93.8
	0.770			105.6
Positive Control Item	0.071	0.063	0.009	9.7	8.7	1.2
	0.053			7.3
	0.066			9.1
Test Item	0.794	0.743	0.044	108.9	101.9	6.1
	0.721			98.9
	0.714			97.9

 

* =          The mean viability of the negative control tissues is set at 100%

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. Cytokine analysis may be useful under certain circumstances e.g. to identify false positive results for skin irritation associated with amine derived chemicals, and therefore the cell culture medium was retained.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 101.9% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 can not be determined).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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.

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control

Value:

41.4

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative control

Value:

0.1

Negative controls validity:

valid

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.

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008, Decarboxylated Rosin (CAS No. 8050 -18 -8) is not classified for skin corrosion/irritation or eye irritation.