Registration Dossier

Administrative data

Description of key information

The aim was to evaluate the possible irritating effects of test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid) after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model) according to OECD 439.

The mean percent viablity of the treated tissues was 94.0 %, versus 1.3% of the positive control (5% Sodium Dodecyl Sulfate). Therefore the test item has to be considered as non-irritant to skin.

Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid) was tested in an vitro eye irritation test according to OECD 438.

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. Therefore the test item needed to be tested additionally in vivo.

The test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid was tested in vivo in rabbits according to OECD Test Guideline No. 405. The ocular reaction observed during the study have been slight to moderate and totally reversible within 72 hours.

In conclusion, the results obtained under the experimental conditions of this study show that the test substance is not irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 - 11 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
Specific details on test material used for the study:
- Lot/batch No.of test material: 05347/MA
- Expiration date of the lot/batch: Retest date June 2022
- Storage condition of test material: room temperature, darkness
- Purity: >99% (GC)
- Purity test date: 31 August 2018
Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed epidermis of normal human keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17
- Tissue batch number: 18-RHE-118
- Production date: cells are grown on inert polycarbonate filters in chemicaly defined medium, for 17 days
- Delivery date: 09 October 2018
- Date of initiation of testing: 09 October 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with 25 x 1 ml of DBPS (Dutscher - Batch No. 4090718)
- Observable damage in the tissue due to washing: rinsed tissues were checked of any coloration and noted to be whitish, comparable coloration to that of the negative control tissues
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader supplied by BioTek (controlled every year and calibrated if necessary) with validated software Gen5 ELISA V1.05.11 supplied by BioTek
- Wavelength: 570 nm
- Filter: no use of filter mentioned
- Linear OD range of spectrophotometer: not mentioned

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.2 (CV = 3.6%), MTT test at 570 nm optical density, specification criteria: OD: > 0.7)
- Barrier function: Exposure time inducing 50% viability using Triton X-100 1% = 4.0 h (Specification criteria: 4.0 <= ET50 <= 10.0 h)
- Morphology: histological observation of HES stained vertical paraffin sections: 7 cell layers (specification criteria: number of cell layers >= 4), absence of significant histological abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: on blood of donor the following was verified: absence of HIV1 and 2 antibodies, absence of hepatits C antibodies, absence of hepatitis B antigen HBsp;
on epidermal cells of the donor the following was verified: absence of mycoplasma
- Reproducibility:within range of historical controls

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- Classification of irritation potential is based upon relative mean tissue viability following the 42-minute exposure period followed by the 42-hour post-exposure incubation period according to:
Relative mean tissue viability is <= 50%: UN GHS Category 2 "irritant" or UN GHS Category 1 "corrosive" in absence of information on a skin corrosion test
Relative mean tissue viability is > 50%: non-irritant UN GHS no category)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 16 μL
- Concentration: undiluted, used as supplied

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Details on study design:
MAIN TEST
Application of test item and rinsing
Exposure: 42 min at room temperature, to ensure a good contact with the epidermises, during all the treatment period, the test and control items were recovered with a nylon mesh provided by Episkin SA
Amount applied: 16 µl
Negative control: 16 µl distilled water
Positive control: 16 µl 5% w/v SDS
Washing: 25 x 1 ml DPBS
Post-exposure incubation: 41 h and 55 min at 37°C

MTT Loading/Formazan Extraction
MTT solution: 300 µl of 1.0 mg/ml per well
Incubation: 3 h at 36.5 and 38 °C
The precipated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was determined by measuring the OD at 570 nm in triplicate, just after dilution of the extracts 1:2 in isopropanol.

DATA EVALUATION
Viability (%): (mean OD test item / OD negative control) x100
The OD values obtained for each test item were used to calculate a percentage of viability relative to the negative control, which was arbitrarely set to 100%.

ACCEPTABILITY
- SD (standard deviation) <= 18%
- Negative control: OD value of the 3 replicates in the range >= 0.8 and <= 3.0, the optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range >=0.4 and <= 1.5 for the negative control.
Irritation / corrosion parameter:
% tissue viability
Value:
94
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.3 %
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none, the rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues
- Direct-MTT reduction:
The direct interaction of MTT with the test item was checked by adding 16 µl of the test item to 300 µl of the solution of MTT at 1 mg/ml (same condition as in the main test). A yellow solution was observed after 3 hours of incubation at approx. 37°C. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
The coloration potential of the test item in water was checked by adding 16 µl of the test item to 300 µl of distilled water. A colourless sollution was obtained after 3 hours of incubation at approx. 37°C. The coloration potential of the test item in isopropanol was checked by adding 16 µl of the test item to 1.5 ml of isopropanol. A colourless solution was obtained after 2 hours of incubation at room temperature. Therefore the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD for the negative control treated tissues was 0.894 and the standard deviation value of the viability was 3.5%
- Acceptance criteria met for positive control: yes, relative mean tissue viability for the positive control treated tissues was 1.3% relative to the negative control treated tissues and the standard deviation value of the viability was 0.1%
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation calculated from individual tissue viabilities of test item treated tissues was 9.3%

Table 1: Individual and average values of OD after 42 minutes exposure

   Well ID  OD  Mean OD/ disc *  Mean OD/product  Viability (%)

Mean viability (%)**

 SD viability   Conclusion
Negative control  SPL1  0.908 / 0.894 / 0.891 0.897   0.894 100.4   100.0 3.5   -
  SPL 2  0.937 / 0.926 / 0.908 0.923    103.3      
  SPL 3  0.867 / 0.840 / 0.878  0.861    96.3      
Positive control  SPL 4  0.011 / 0.014 / 0.013   0.012  0.012  1.3 1.3  0.1  irritant
  SPL 5  0.012 / 0.011 / 0.011  0.011    1.2      
  SPL 6 0.013 / 0.013 / 0.014  0.013    1.5      
Test item  SPL 7 0.917 / 0.939 / 0.941  0.932  0.840 104.3  94  9.3  non irritant
   SPL 8  0.772 / 0.764 / 0.775  0.770   86.2       
  SPL 9  0.816 / 0.832 / 0.807  0.818   91.5      

 * = mean of 3 values (triplicate of the same extract)

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

OD: optical density

SPL: sample

SD: standard deviation

Interpretation of results:
GHS criteria not met
Conclusions:
The mean percent viablity of the treated tissues was 94.0 %, versus 1.3% of the positive control (5% Sodium Dodecyl Sulfate). The test item has to be considered as non-irritant to skin.
Executive summary:

The aim was to evaluate the possible irritating effects of test item Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model).

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 16 µL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours and 55 minutes postincubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.  The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 28 July 2015 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The mean percent viability of the treated tissues was 94.0%, versus 1.3 % in the positive control (5% Sodium Dodecyl Sulfate).  

In accordance with the Regulation EC No. 1272/2008, the test item Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.  No hazard statement or signal word is required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
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. certificate)
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
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
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
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.
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
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
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.


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

 

 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 

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.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
October 9th, 2017
Deviations:
yes
Remarks:
Due to animal welfare reasons analgesia was uses, for details see section "Any other information on materials and methods incl. tables" below
GLP compliance:
yes (incl. certificate)
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by 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
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Granja San Bernardo (Tulebras, Navarra - Spain)
- Age at study initiation: 9, 10 or 11 weeks old
- Housing: single housing, animals were provided with environmental enrichment (Aspen brick)
- Diet: ENVIGO - 2030 C, ad libitum
- Water: Tap water from public distribution system ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 to 70
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 ml
Duration of treatment / exposure:
single administration, in each animal the test article was introduced into the conjunctival sac of one eye after gently pulling the lower lid away from the eyeball, afterwards the eyelid was closed for 1 second, unlimited exposure (not rinsed).
Observation period (in vivo):
1 hour, 24, 48 and 72 hours following treatment.
Number of animals or in vitro replicates:
3 male
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM: acc. to guideline
Irritation parameter:
iris score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
chemosis score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Remarks:
animal #1
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
animal #2
Basis:
mean
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 48 h
Irritation parameter:
iris score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
cornea opacity score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
0.7
Max. score:
1
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
animal #3
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Irritant / corrosive response data:
At the conjuncutivae level: a slight to moderate redness noted 1 hour after the test item instillation in all animals and totally reversible between Days 1 and 2. This reaction was associated with a slight chemosis noted 1 hour after treatment in all animals and totally reversible between Days 1 and 2. At the cornea level: a slight opacity noted 24 hours after treatment in two animals and totally reversible between Days 2 and 3.
Other effects:
none

Table 1: Irritant/corrosive response data at each observation time

Score at time point / Reversibility

 

Cornea

Iris

Conjunctivae Redness

Conjunctiva Chemosis

1 h

0/0/0

0/0/0

2/2/2

1/1/1

24 h

0/1/1

0/0/0

0/2/1

0/1/1

48 h 0/0/1 0/0/0 0/0/0 0/0/0
72 h 0/0/0 0/0/0 0/0/0 0/0/0
 Average 24, 48, 72 h

0.33

0

0.33

0..22

 Reversibility *

c.

n.a.

 c.

 c.

 

* Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible, n.a.= not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the result of this study, the substance Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid is not irritating to the eyes.
Executive summary:

The test item Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid was instilled, as supplied, into the eye of three New Zeland rabbits at the dose of 0.1 ml. The experimental protocol was established on the basis of the official method as defined in the OECD Test Guideline No. 405 dated October 9th, 2017 and performed in accordance with the Principles of GLP. The ocular reaction observed during the study have been slight to moderate and totally reversible within 72 hours.

In conclusion, the results obtained under the experimental conditions of this study show that the test substance is not irritating to the eyes. In accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures, no symbol and warning label are required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid)

was non-irritant to skin in an in vitro test according to OECD 439.

Supporting the source substance 1,2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters was in an in vivo test only slightly irritating to the skin, the effects in the skin were fully reversible within 8 days.

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

(Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid) was tested in an in vitro eye irritation test according to OECD 438.

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. Therefore the test item needed to be tested additionally in vivo.

The test item Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid) was instilled, as supplied, into the eye of three New Zeland rabbits at the dose of 0.1 ml. The experimental protocol was established on the basis of the official method as defined in the OECD Test Guideline No. 405 dated October 9th, 2017 and performed in accordance with the Principles of GLP. The ocular reaction observed during the study have been slight to moderate and totally reversible within 72 hours.

In conclusion, the results obtained under the experimental conditions of this study show that the test substance is not irritating to the eyes. In accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures, no symbol and warning label are required.

Supporting is the outcome of the in vivo eye irritation study of the read-across substance 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester.

Justification for classification or non-classification

The results of the skin and eye irritating studies show that Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohols, C24-26,branched and cyclic with 1,2,4-benzenetricarboxylic acid) does not warrant classification according to Regulation (EC) No 1272/2008.