Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15th of October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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:

adopted 25 June 2018

Deviations:

yes

Remarks:

Deviation is considered as without impact on the results of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2017/33, dated 27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1)

EC Number:

949-745-1

Molecular formula:

not applicable UVCB

IUPAC Name:

Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1)

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, Batch no.: 05347/MA
- Expiration date of the lot/batch: Retest date June 2022
- Purity test date: 31 August 2018
- Storage condition of test material: room temperature, darkness

Test animals / tissue source

Species:

other: isolated chicken eye

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse Etablissement Brun, 33820 Etauliers, France, eyes collected from chicken killed for human consumption
- Number of animals: not mentioned
- Characteristics of donor animals: approx. 7 weeks old, 1.5 - 2.5 kg, sex not mentioned
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. Eyes were enucleated at test laboratory and transferred to a chamber of the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip in the range of 0.1 to 0.15 ml/min. at temperatures between 32.3 and 32.4 °C.
- Indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: not mentioned

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µl

Duration of treatment / exposure:

10 seconds, then rinsed from the eye with 20 ml of physiological saline at ambient temperature

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated at 30, 75, 120, 180, and 240 minutes after post-treatment rinse

Number of animals or in vitro replicates:

3 replicates for the test item, 3 replicates for the positive control, 1 replicate for the negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, 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 was 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. 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 postioned in the superfusion apparatus such that 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 32.3 and 32.4°C. 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 procedur. 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 the 10% from the mean value for all eyes were also rejected.

EQUILIBRATION AND BASELINE RECORDINGS
All examined and approved eyes were incubated between 45 and 64 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 baseline. The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
3 replicates for the test item, 3 replicates for the posisitve control, 1 replicate for the negative control

NEGATIVE CONTROL USED
yes, physiological saline

POSITIVE CONTROL USED
yes, Benzalkonium chloride, 5% in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 µL of the test item was applied, as supplied, to the cornea such that the entire surface of the cornea was evenly covered with the test item. The test item was applied for 10 seconds and then rinsed from the eye with 20 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treament rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed from the eye with 20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline : The eyes were incubated between 45 and 64 minutes instead of between 45 and 60 minutes, as initially scheduled. As the results obtained with the eyes treated with the negative control which has the longest duration of acclimation were conformed to what expected, this deviation is considered as without impact on the results of the study.

METHODS FOR MEASURED ENDPOINTS:
All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness the slit-width was set at 9 1/2, equally 0.095 mm. Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity as observed at any time point, an overall category score was then given for each test or control item. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g. pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention were determined at each of the above time points. Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope and was expressed as a percentage. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item. The mean fluorescein retention value for all test eyes 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. Morphological effects 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 to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%) : according to guideline
- Mean maximum opacity score : according to guideline
- Mean fluorescein retention score at 30 minutes post-treatment ; according to guideline

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

maximal mean score

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class II

Results 2

Irritation parameter:

fluorescein retention score

Remarks:

mean score

Value:

3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class IV

Results 3

Irritation parameter:

percent corneal swelling

Remarks:

maximal mean swelling

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class I

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: At all examination time points no morphological effects were noted in the negative control and the test item treated eyes. The eyes treated with the positive control showed blisters on the cornea from 30 minutes post-dose in all 3 tested eyes.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The combination of the three endpoint for the negative control (physiological saline) was 3 x I, therefore the negative control is classified as "No category", as expected.
The combination of the three endpoints for the positive control (5% Benzalkonium chloride) was 2 x IV, 1 x III, therefore the positive control is classified as "Corrosive/Severe Irritant", as expected.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not different

The ocular reactions observed in eyes treated with the test item were: - maximal mean score of corneal opacity: 1.0, corresponding to ICE class II; - mean score of fluorescein retention: 3.0, corresponding to ICE class IV; - maximal mean corneal swelling: 2%, corresponding to ICE class I.

The combination of the three endpoints for the test ISOFOL ESTER 4693 (Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid) was 1 x IV, 1 x II, 1 x I.

A test item that is not predicted as causing serious eye damage with the ICE test method would require additional testing (in vitro and/or in vivo) to establish a definitive classification.

Any other information on results incl. tables

Table #1: Values for Evaluation of Corneal Lesions after Treatment with Test Item

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	4	0	1	1	1	1	1
	5	0	1	1	1	1	1
	6	0	1	1	1	1	1
Mean		0.0	1.0	1.0	1.0	1.0	1.0
ICE class		II
Fluorescein retention	4	0.5	3	-	-	-	-
	5	0.5	3	-	-	-	-
	6	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class		IV		-	-	-	-
Corneal thickness	4	0.60	0.60	0.62	0.62	0.62	0.62
	5	0.58	0.58	0.58	0.58	0.58	0.58
	6	0.58	0.58	0.58	0.58	0.58	0.58
Corneal swelling (%)	4	-	0	3	3	3	3
	5	-	2	2	2	2	2
	6	-	2	2	2	2	2
Mean		-	1	2	2	2	2
ICE class		I
Combination of 3 endpoints		1 x IV, 1 x II, 1 x I
Classification		no prediction can be made

Table #2: Values for Evaluation of Corneal Lesions after Treatment with Positive Control (5% (w/v) Benzalkonium chloride

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	1	0	3	3	3	3	3
	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
Fluorescein retention	1	0.5	3	-	-	-	-
	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class		IV		-	-	-	-
Corneal thickness	1	0.58	0.65	0.68	0.72	0.75	0.78
	2	0.60	0.68	0.74	0.76	0.79	0.79
	3	0.61	0.63	0.69	0.70	0.78	0.78
Corneal swelling (%)	1	-	12	17	24	29	34
	2	-	13	23	27	32	32
	3	-	3	13	15	28	28
Mean		-	10	18	22	30	31
ICE class		III
Combination of 3 endpoints		2 x IV, 1 x III
Classification		Category 1: Corrosive / Severe irritant

Table #3: Values for Evaluation of Corneal Lesions after Treatment with Negative Control (Physiological Saline)

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	16	0	0	0	0	0	0
ICE class		I
Fluorescein retention	16	0	0	-	-	-	-
ICE class		I		-	-	-	-
Corneal thickness	16	0.58	0.58	0.58	0.58	0.58	0.58
Corneal swelling (%)	16	-	0	0	0	0	0
ICE class		I
Combination of 3 endpoints		3 x I
Classification		No category

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The results obtained under these experimental conditions lead to the category "no prediction can be made", as defined in the guideline OECD No. 438, therefore the test item is not predicted as causing serious eye damage (Category I) or as not classified for eye irritation/serious eye damage (No Category) with the Isolated Chicken Eye test method.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid 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 and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.  The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 438 adopted 25 June 2018 and the test method B.48 – Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The ocular reactions observed in eyes treated with the test item were: - maximal mean score of corneal opacity: 1.0, corresponding to ICE class II; - mean score of fluorescein retention: 3.0, corresponding to ICE class IV; - maximal mean corneal swelling: 2%, corresponding to ICE class I.

The combination of the three endpoints for the test ISOFOL ESTER 4693 (Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid) was               1 x IV, 1 x II, 1 x I.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was  2 x IV, 1 x III. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

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.

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) is required to establish a definitive classification.