Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 2nd, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

October 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of Isoamyl salicylate and 2-Methylbutyl salicylate

IUPAC Name:

Reaction Mass of Isoamyl salicylate and 2-Methylbutyl salicylate

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Etablissement Brun, 33820 Etauliers, France), the chickens were killed for human consumption
- Characteristics of donor animals: spring chickens (approximately 7 weeks old, 1.5 - 2.5 kg)
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The head collection was at 8:30 am and arrived at 10:15 am at the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

No treatment incubation is performed.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Dissection of the eyes: the eyelids were carefully excised and the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
Selection of the eyes: The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.1ºC and 32.3ºC. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of >0.5; (ii) corneal opacity>0.5, or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
The eyes examined and approved were incubated between 45 and 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e. Time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 30 μL Physiological saline - Dutscher Batch No. 3013132 (1 replicate)

POSITIVE CONTROL USED: 30 μL 5% (w/v) Benzalkonium chloride - Sigma Batch No. BCBQ9761V (3 replicates)

APPLICATION DOSE AND EXPOSURE TIME
30 μL of the test item were applied for 10 seconds

OBSERVATION PERIOD
Pretreatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was measured using a HaagStreit BP900 slit-lamp microscope with depth measuring device no. I. It was calculated using the area of the cornea that was most densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: It was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: It was measured using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I, the slit-width was set at 91/2, equalling 0.095 mm. The mean percentage of corneal swelling was calculated for all observation time points.
- Macroscopic morphological damage to the surface: include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. The classification of these findings is subjective according to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%): (corneal thickness at time t -corneal thickness at time=0/corneal thickness at time=0)x100.
- Mean maximum opacity score: 0= No opacity; 0.5= Very faint opacity; 1= Scattered or diffuse areas, details of the iris clearly visible; 2= Easily discernible translucent area, details of the iris are slightly obscured; 3= Severe corneal opacity, no specific details of the iris are visible, size of the pupil is barely discernible; 4= Complete corneal opacity, iris invisible.
- Mean fluorescein retention score at 30 minutes post-treatment: 0= No fluorescein retention; 0.5= Very minor single cell staining; 1= Single cell staining scattered throughout the treated area of the cornea; 2= Focal or confluent dense single cell staining; 3= Confluent large areas of the cornea retaining fluorescein.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. All endpoints were evaluated separately to generate an ICE class for each endpoint and then combined to generate an Irritancy Classification for the test item. The ICE classes were assigned based on a predetermined range.
ICE classification criteria for corneal thickness: 0 to 5 = class I; >5 to 12= class II; >12 to 18 (>75 min after treatment)=class II; >12 to 18 (≤75 min after treatment)=class III;>18 to 26= class III; >26 to 32 (>75 min after treatment)= class III; >26 to 32 (≤75 min after treatment)= class IV; >32=class IV. ICE classification criteria for opacy: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-4.0=class IV.
ICE classification criteria for mean fluorescein retention: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-3.0=class IV.
Overall in vitro classifications:
No category= 3 x I / 2 x I, 1 x II
No prediction can be made= Other combinations
Category 1 (Corrosive/Severe Irritant)= 3 x IV / 2 x IV, 1 x III / 2 x IV, 1 x II / 2 x IV, 1 x I / Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) / Corneal opacity

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Maximal Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Results 2

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class IV

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximal mean

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The test is considered accpetable if the negative control is identified as GHS Non-Classified, The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The test is considered accpetable if the positive control is identified as GHS Category 1. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
- Range of historical values if different from the ones specified in the test guideline: Regular positive controls performed during 2017 had a combination of endpoints 3 x IV, and would be classiied as corrosive, severe irritant. The negative control combinations of endpoints ranged from 2 x I, 1 x II to 3 x I and were all classified as GHS no category.

Any other information on results incl. tables

Table 1. Individual and average values for evaluation of corneal lesions after treatment with the negative control (Physiological saline)

Endpoint measured	Eye No.	0	30	Time (min) 75	120	180	240
Corneal opacity	16	0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I
Fluorescein retention	16	0	0	-	-	-	-
ICE class			I	-	-	-	-
Corneal thickness	16	0.57	0.57	0.57	0.57	0.57	0.57
Corneal swelling (%)	16	-	0	0	0	0	0
ICE class			I
Combination of the 3 Endpoints		3 x I
CLASSIFICATION		No category

Note: No morphological effects were noted, whatever the examination time.

Table 2. Individual and average values for evaluation of corneal lesions after treatment with the positive control ( 5% (w/v) Benzalkonium chloride)

Endpoint measured	Eye No.	0	30	Time (min) 75	120	180	240
	1	0	3	3	3	3	3
Corneal opacity	2	0	3	3	3	3	3
	3	0	3	3	3	3	3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
ICE class			IV
	1	0.5	3	-	-	-	-
Fluorescein retention	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
	1	0.56	0.66	0.71	0.80	0.82	0.84
Corneal thickness	2	0.60	0.71	0.78	0.82	0.82	0.83
	3	0.61	0.72	0.76	0.84	0.85	0.86
	1	-	18	27	43	46	50
Corneal swelling (%)	2	-	18	30	37	37	38
	3	-	18	25	38	39	41
Mean		-	18	27	39	41	43
ICE class			IV
Combination of the 3 Endpoints		3 x IV
CLASSIFICATION		Category 1 : Corrosive / Severe irritant

Blisters on the cornea noted 30 minutes post dose in eyes No.1, 2, 3.

Table 3. Individual and average values for evaluation of corneal lesions after treatment with the test item.

Endpoint measured	Eye No.	0	30	Time (min) 75	120	180	240
	7	0	0	0	0	0	0
Corneal opacity	8	0	0	0	0	0	0
	9	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I
	7	0.5	3	-	-	-	-
Fluorescein retention	8	0.5	3	-	-	-	-
	9	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
	7	0.57	0.63	0.64	0.64	0.64	0.66
Corneal thickness	8	0.58	0.62	0.62	0.62	0.62	0.64
	9	0.60	0.63	0.63	0.63	0.63	0.63
	7	-	11	12	12	12	16
Corneal swelling	8	-	7	7	7	7	10
(%)	9	-	5	5	5	5	5
Mean		-	7	8	8	8	10
ICE class			II
Combination of the 3 Endpoints		1 x IV, 1 x II, 1 x I
CLASSIFICATION		No prediction can be made

Note: No morphological effects were noted, whatever the examination time.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The combination of the 3 endpoints fot the test item was 1 x IV, 1x II, 1 x I, according to OECD 438. Therefore, no prediction can be made.

Executive summary:

The eye irritation of the test item has been evaluated using the Isolated Chicken Eye test method, according to OECD 438 and EU method B.48, under GLP conditions. The test item was applied, as supplied, at the dose of 30 μL to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control ( 5% (w/v) Benzalkonium chloride) and one eye with a negative control (physiological saline). Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention and were evaluated at pretreatment and at 30, 75, 120, 180 and 240 minutes post-dose. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Positive control and negative control results were valid. The mean corneal opacity for the test item treated corneas was 0, corresponding to ICE category class I; the mean score of fluorescein retention was 3, corresponding to ICE category class IV; and the mean corneal swelling was 10%, corresponding to ICE class II. The combination of the 3 endpoints for the test item was 1 x IV, 1x II, 1 x I, according to OECD 438. Therefore, no prediction can be made.