Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

Endpoint summary

Description of key information

Skin:

REACH_not corrosive | Human Skin Model | OECD 431 | #key study#

REACH_not irritating | Human Skin Model | ECVAM Validation Study | #key study#

Eye:

REACH_not irritating | HET-CAM | #key study#

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:

June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 431 (In vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiDerm Skin Model (EPI-200)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

Recommended test system in international guldelines (OECD and EC)

Vehicle:

water

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 10526 klt H).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, hlghly differentiated model of the human epidermis. It consists of organlzed basal, spinous and granular layers, and a multi-Iayered stratum eomeum containing intercellular lamellar lipid layers arranged In patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm2) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg with 25 µl water

Duration of treatment / exposure:

3 min., 1 h

Number of replicates:

two tissues for a 3 min exposure
two tissues for a 1 h exposure

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min. exposure

Value:

108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure

Value:

108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

no other effects

Mean tissue viability in the in vitro skin corrosion test with the Substance

	3 min application viability (percentage of control)	1 hr application viability (percentage of control)
Negative control	100	100
Test Substance	108	108
Positive control	5	4

The Table shows the mean tissue viability obtained after 3 minutes and 1 hour treatment with the Substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with the Substance compared to the negative control tissues was 108% at both exposure times. Since the mean relative tissue viability for the Substance was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the Substance is considered to be not corrosive.

The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability of the 3 minutes exposure of the positive control was 5%. The maximum inter tissue variability in viability between two tissues treated identically was less than 21% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 12%. It was therefore concluded that the test system functioned properly.

Interpretation of results:

other: not corrosive

Conclusions:

It is concluded that this test is valid and that the Substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

This report describes the corrosive properties of the Substance on a human threedimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the Substance was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines.

The Substance was a very viscous amber liquid. Since the Substance was very viscous it was applied like a solid test substance. Approximately 25 mg of the Substance with 25 μl of Milli-Q water was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability after 3 minutes exposure of 5%. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter tissue variability in viability between two tissues treated identically was less than 21% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 12%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.

The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with the Substance compared to the negative control tissues was 108% at both exposure times. Since the mean relative tissue viability for the test substance was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the Substance is considered to be not corrosive.

Finally, it is concluded that this test is valid and that the Substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure according to validation study and GLP

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study

Principles of method if other than guideline:

- L’Oréal Standard Operating Procedure, ECVAM Skin Irritation Validation Study, Validation of the Episkin Skin Irritation Test (42 hours) assay for the prediction of acute skin irritation of chemicals, January 2005.
- The ESAC statement (ECVAM Scientific Advisory Committee) on the validity of in vitro tests for skin irritation, April 2007

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiSkin Standard Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

not specified

Justification for test system used:

Recommended test system by the Institute for Health & Consumer Protection, European Centre for the Validation of Alternative Methods (ECVAM). The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test substance by assessment of its effect on a three dimensional human epidermis model.

Vehicle:

water

Details on test system:

EPISKIN Standard Model™ (EPISKIN-SM™, 0.38 cm2, Lot no.: 09-EKIN-001).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13-days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg with 5 µl water

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 h (incubation period)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Time point: 15 min.

Value:

75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

no other effects

Mean absorption in the in vitro skin irritation test with the Substance

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.769	0.765	0.768	0.767	±	0.002
Tested Substance	0.574	0.555	0.594	0.574	±	0.019
Positive control	0.023	0.031	0.025	0.026	±	0.004

OD = optical density / SD = Standard deviation / Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (mean OD₅₇₀= 0.000). Isopropanol was used to measure the background absorption.

Mean tissue viability in the in vitro skin irritation test with the Substance 

	Mean tissue viability (percentage of control)  ±SD
Negative control	100± 0.27
Tested Substance	75± 2.51
Positive control	3± 0.52

 

The Table shows the mean tissue viability obtained after 15 minutes treatment with the Substance compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the Substance compared to the negative control tissues was 75%. Since the mean relative tissue viability for the Substance was above 50%. The Substance is considered to be non-irritant.

 

The positive control had a mean cell viability after 15 minutes exposure of 3%. The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was ≥0.6. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. 

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that the Substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

This report describes the ability of the Substance to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM^TM)). The possible skin irritation potential of the Substance was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent Skin irritation documents.

Batch NB2890-61 of the Substance was a very viscous amber liquid. Skin tissue was moistened with 5 µl of MIlli-Q water and 10 mg of the Substance was applied directly on top of the skin tissue. After a 42 hours incubation period determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the Substance compared to the negative control tissues was 75%. Since the mean relative tissue viability for the Substance was above 50% after 15 minutes treatment the Substance is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 3%. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was ≥0.6. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that the Substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Reference 3

Endpoint:

skin irritation: in vivo

Remarks:

The test was performed for registration purpose outside EU

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June-July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Ministry of Environmental Protection of People's Republic of China in the year of 2013.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: Japanese White

Details on test animals or test system and environmental conditions:

ANIMALS
- Number: 3 animals
- Sex: Male
- Age: Young adult, 12 weeks at arrival and 13 weeks at dosing.
- Body Weight Range: 1827.0-2012.7 g at receipt and 2052.4-2170.4 g at dosing.
- Physical Check-up and Acclimatization: Physical check-up was made to all animals on arrival. Healthy young adult animals were acclimatized to the laboratory
conditions and housed individually for 10-11 days prior to dosing, during which clinical observations were performed daily to ensure that all animals showed no abnormalities.
All animals were weighed and marked by the special animal markers beginning from No. 1100 to 1102 and number written on cage cards within 24 hours after arrival.

HUSBANDRY
Temperature and humidity were controlled automatically and recorded daily. The temperature was within 17-23°C and relative humidity was within 40-70%. The
lighting sequence was 12 hours light, 12 hours dark.
The animals were individually housed in suspended, stainless steel cages (L50cm x W40cm x H40cm) on cage racks during the study. They were provided with pellet breeding rabbit diet with complete nutrition supplied by Beijing Keaoxieli Feed Co., Ltd. [Product License No: SCXK(Jing) 2014-0010, Product batch No.: 17064211]. All nutrition components and contaminants were within the permitted limits described in the national standard (GB14924.3-2010 and GB14924.2-2001). Drinking water was purified by the HT-R01000 purity system, water analysis was conducted routinely (annually), and all parameters were within the permitted limits described in the national standard (GB5749 2006). Diet and drinking water were considered not to contain any contaminants that could
reasonably be expected to affect the result, purpose and integrity of the study. Diet and drinking water were available to the animals ad libitum during the test.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

6 d

Number of animals:

3

Details on study design:

To investigate if the test material has dermal irritation potential, an in vivo test in rabbits was conducted in a sequential way.
Initial Test: The test was performed initially using one animal with one control patch and one test patch for an exposure period of 4 hours.
Confirmatory Test: Corrosive or severely irritant effects of the test item were not observed in the initial test within 24 h after patch removal, a confirmatory test was conducted by exposing two additional animals simultaneously. Each animal received application of one control patch and one test patch for an exposure period of 4 hours.

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

1100

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

1100

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

1101

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 6 d

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

1101

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

1102

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 6 d

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

1102

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible within: 48 h

Body weight

The animals showed expected gains in body weights during the course of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters was considered to be slightly irritating to rabbit skin; no classification is warranted.

Executive summary:

The study was performed to assess the acute dermal irritation/corrosion of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in J apanese White rabbits. The method was designed to be in accordance with the Guidelines for the testing of chemieals "Acute Dermal Irritation/Corrosion Test" (TG 404) published by the Ministry of Envirenmental Protection of People's Republic of China in the year of 2013.

Method: Three male rabbits were used for the study. A quantity of 0.5 mL of the test item was applied to the right side of back skin of each animal for an exposure period of 4 hours. The untreated skin on the left back area of the animal served as the control. Sighting of clinical signs were performed once daily throughout the study. Observation of the skin sites of each animal for signs of erythema/eschar and oedema were all recorded immediately, and at approximately 1, 24, 48 and 72 hours after patch removal, and all local toxic effects of skin were also fully described and recorded. Dermal reactions (erythema/eschar and oedema) of test sites were scored at approximately 1, 24, 48 and 72 hours after patch removal. Mean scores of erythema/eschar or oedema at 24, 48 and 72 hours were calculated for the tested skins after patch removal Individual animal body weights were recorded on the day of dosing and on the completion of final observations.

Results:

- Clinical Observations: No abnormal signs or symptoms were observed in all the three animals throughout the course of the study.

- Skin Reactions Examination: There were no observable abnormalities for the three control sites in skin reaction examinations at all observation intervals. Animal # 1100 showed no observable abnormalities for the test site in the skin reaction examination at 0, 1, 24, 48 and 72 hours after patch removal. Animal # 1101 showed very slight erythema (barely perceptible) at 0, 1, 24, 48, 72 hours and day 4 and 5 after removal of the test item patch. Animal # 1102 showed very slight erythema (barely perceptible) at 0, 1, 24, 48, 72 hours and day 4 and 5 of the observation; very slight oedema (barely perceptible) at 0, 1 and 24 hours after removal of the test item patch.

- Grading of Skin Reactions: The mean scores of erythema/eschar and oedemas for animal # 1100 at the 24, 48 and 72-hour observation points were all 0 after patch removal ofthe test item. The mean scores of erythema/eschar and oedemas for animal # 1101 at the 24,48 and 72-hour observation points were 1 and 0, respectively, after patch removal of the test item. The mean scores of erythema/eschar and oedemas for animal # 1102 at the 24, 48 and 72-hour observation points were 1.0 and 0.33, respectively, after patch removal of the test item.

- Body Weights: All animals showed expected gains in body weights during the course of study.

Conclusion: Based on the results of this study, Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters was considered to be slightly irritating to rabbit skin; no classification is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure according to validation study and GLP

Qualifier:

according to guideline

Guideline:

other: HET-CAM Test

Version / remarks:

INVITTOX protocol 47 / The HET -CAM test - Method of Spielmann and Liebsch, January 1992.

Principles of method if other than guideline:

The aim of this study was to evaluate the ocular irritancy of the Substance as measured by its ability to induce adverse changes in the chorioallantoic membrane (CAM) of chicken eggs. The study procedures were based on the following documents and follow international recommendations
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) test method, March 2006.
- INVITTOX protocol 47. The HET-CAM test - Method of Spielmann and Liebsch, January 1992.

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: white leghorn

Details on test animals or tissues and environmental conditions:

Test System: Fresh, fertilised white leghorn chicken eggs weighing between 53 and 62 grams.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

300 mg test item
0.3 mL positive control (1% sodium dodecylphosphate prepared in physiological saline) and negative control (physiological saline)

Duration of treatment / exposure:

20 seconds

Observation period (in vivo):

5 minutes

Number of animals or in vitro replicates:

3

Details on study design:

The number of eggs used in the study was 3 per treatment group. On day 9 of incubation the air cells of the eggs were marked and cut off with a rotating saw blade. The uncovered inner membrane was moistened with physiological saline and subsequently the egg was kept at room temperature. Subsequently the physiological saline was removed and the inner egg membrane carefully eliminated using forceps to expose the CAM.
Three eggs were treated for 20 seconds with 300 mg of the Substance or with 0.3 mL of the negative or positive control solutions. After the treatment period the eggs were carefully rinsed with approximately 5 mL of physiological saline. The appearance of haemorrhage, vessel lysis and coagulation on the CAM was monitored and recorded over the following 280-second period.

Results from the three test method endpoints were evaluated separately for each egg. The irritancy score (IS) was calculated using the observed haemorrhage time, lysis time and coagulation time.

Irritation parameter:

in vitro irritation score

Remarks:

HET-CAM

Run / experiment:

20 sec treatement, 280 sec observation

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

score 0

Positive controls validity:

valid

Remarks:

score 14.7

Other effects / acceptance of results:

No other effects observed.
The mean in vitro irritancy score obtained with Ihe negative control was less than 1 indicating that the negative control did not induce irritancy on the chorioallantoic membrane.
The mean in vitro irritancy score of the positive control (1 % (wlv) Sodium dodecyl sulphate) was within the historical control data range. It was therefore concluded that the test conditions were adequate and that Ihe lest system functioned properly.

The results from the three test method endpoints and irritancy scores of the individual eggs.  Egg Heamorrhage time (sec)  Lysis time (sec) Coagulation time (sec) irritancy score (IS)  Mean irritancy score (IS)                 Negative control  1 301  301  301  0.0     2 301  301  301  0.0     3 301  301  301  0.0               0.0                 1 % Sodium dodecyl sulphate  5 301  30  20  14.8     6 301  30  20  14.8     7 301  35  20  14.6               14.7                Test Substance  15 301  301  301  0.0     16 301  301  301  0.0     17 301  301  301  0.0               0.0 Note: 301 seconds was noted when no effect was oberved after 5 minutes of observation.

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: ICCVAM and NICEATM

Conclusions:

It is concluded that this test is valid and that the substance is not irritant in the Hen’s Egg Test – Chorioallantoic Membrane Test under the experimental conditions described in this report.

Executive summary:

The mean in vitro irritancy score obtained with the negative control was less than 1 indicating that the negative control did not induce irritancy on the chorioallantoic membrane. The mean in vitro irritancy score of the positive control (1% (w/v) Sodium dodecyl sulphate) was within the historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The mean in vitro irritancy score obtained during a 280-second observation period after 20 seconds treatment with the Substance was 0.

Finally, it is concluded that this test is valid and that the Substance is not irritant in the Hen’s Egg Test – Chorioallantoic Membrane Test under the experimental conditions described in this report.

This report describes the ocular irritation properties of the Substance on the chorioallantoic membrane of chicken eggs. The possible ocular irritancy of teh Substance was tested through topical application for 20 seconds.

The study procedures described in this report were based on recent HET-CAM documents.

The substance was a very viscous amber liquid. The test substance was applied undiluted (300 mg) directly on top of the chorioallantoic membrane.

The mean in vitro irritancy score obtained with the negative control was less than 1 indicating that the negative control did not induce irritancy on the chorioallantoic membrane. The mean in vitro irritancy score of the positive control (1% (w/v) Sodium dodecyl sulphate) was 14.7 and within the historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The mean in vitro irritancy score obtained during a 280-second observation period after 20 seconds treatment with the Substance was 0.

Finally, it is concluded that this test is valid and that the Substance is not irritant in the Hen’s Egg Test – Chorioallantoic Membrane Test under the experimental conditions described in this report.

Reference 2

Endpoint:

eye irritation: in vivo

Remarks:

The test was performed for registration purpose outside EU

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June - July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

"Acute Eye lrritation/Corrosion Test" (TG 405) published by the Ministry of Environmental Protection of People's Republic of China in the year of 2013.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: Japanese White

Details on test animals or tissues and environmental conditions:

ANIMALS
- Species: Albino rabbit
- Strain: Japanese White
- Number: 3
- Sex: Male
- Age: Young adult, 84 days at receipt and 89-90 days at dosing.
- Body Weight Range: 2017.1-2324.9 g at receipt and 2218.2-2563.3 g at dosing
- Physical Check-up and Acclimatization: Physical check-up was made to all animals on arrival. Healthy young adult animals were acclimatized to the laboratory conditions and housed individually for 5-6 days prior to dosing, during which clinical observations were performed daily and all animals showed no abnormality. All animals were weighed and marked by the special animal markers beginning from No. 1100 to 1102 and number written on cage cards within 24 hours after arrival.

HUSBANDRY
Animals were individually housed in suspended, stainless wire cages (W40cmxL50cmxH40cm) on cage racks during the study.
Temperature and humidity were controlled automatically and daily recorded. The temperature was within 17-23 ° C and relative humidity was within 40-70%. The lighting sequence was 12 hours light, 12 hours dark.
Animals were provided with pellet breeding rabbit diet with complete nutrition supplied by Beijing Keaoxieli Feed Co., Ltd. (Product License No: SCXK (Jing)
2014-0010, Product batch No.: 17064211). Analysis reports of diet were supplied by the supplier. All the nutrition components and contaminants were within the permitted limits described in the national standard (GB14924.3-2010 and GB14924.2-2001).
Drinking water was purified by the HT-R01000 purity system. Water analysis was conducted routinely (annually), and all parameters were within the permitted limits described in the national standard (GB5749-2006).
Diet and drinking water were considered not to contain any contaminants that could reasonably be expected to affect the result, purpose and integrity of the study. Diet and drinking water were available to the animals ad libitum during the test.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

eyes unrinsed

Observation period (in vivo):

1, 24, 48 and 72 hours after administration

Number of animals or in vitro replicates:

3

Details on study design:

To investigate if the test material had eye-irritation potential, an in vivo test in rabbits was conducted in a sequential way.
Initial Test: Testing was initially conducted in one animal.
Confirmatory Test: A corrosive or severe irritant effect was not observed in the initial test. The confirmatory test was conducted using two additional animals simultaneously.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible

Other effects:

No adverse systemic effects were observed in any animal during the study.
All animals showed expected bodyweight gains during the study.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters was not irritating on rabbit eyes. No classification is warranted according to GHS classification criteria for the eye irritation.

Executive summary:

The study was performed to assess the acute eye irritation/corrosion of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in rabbits. The method was designed to be in accordance with the Guidelines for the testing of chemicals "Acute Eye lrritation/Corrosion Test" (TG 405) published by the Ministry of Envirenmental Protection of People's Republic of China in the year of 2013.

Method

Three male rabbits were used for the study. Each anirnal was administered with 0.1 mL of the test item in to the right eye. The untreated left eye served as the control. Immediately after administration of the test item, the initial reaction was observed and recorded. Test anirnals were routinely observed and recorded for clinical signs of pain and/or distress and any adverse systemic effects twice daily for

3 days after administration of the test item, with a minirnum of 6 hours between observations.

Both eyes of the three anirnals were examined by a binocular loupe at approxirnately 1, 24, 48 and 72 hours after administration. The ocular reactions and any other lesions in the eye were recorded at each examination. Fluorescein stain examination was used at approxirnately 24 hours after administration. Ocular lesions (cornea, iris, conjunctivae redness and chemosis) for the treated eye of each anirnal were scored

and recorded at each examination. Mean scores of ocular lesions at approxirnately 24, 48 and 72 hours after administration were calculated for the treated eyes. Individual anirnal body weights were recorded on the day of dosing and on the completion of the final observations.

Results

- Clinical Observations: No adverse systemic effects were observed in any anirnals during the study.

- Eye Examination: There were no abnormalities for all eyes in eye examination approximately 24 hours prior to dosing. There were no observable abnormalities in eye examinations for the three control eyes at all observation intervals after administrations. There were no observable abnormalities in eye examinations for the three treated eyes at all observation intervals after administrations.

- Fluorescein Examination: There was no retention of fluorescein for the three treated and control eyes approximately 24 hours prior to dosing.

All of the three treated eyes (3/3) showed no retention of fluorescein at approximately 24 hours after administrations.

- Eye Reaction Scoring: Individual scores for cornea, opacity, iris, conjunctivae redness and conjunctivae swelling of the three treated eyes (from 1100 to 1102) were all 0 at 1, 24, 48 and 72 hours after administrations. Mean scores for cornea opacity, iris, conjunctivae redness and conjunctivae swelling of the three treated eyes (from 1100 to 1102) were all 0.

- Body Weights: All animals showed expected gains in body weights during the study.

Conclusion

Under the conditions of this study, Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters was not irritating on rabbit eyes. No classification is warranted according to GHS classification criteria for the eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The test substance did not produce skin irritation and eye irritation. The substance does not need to be classified for irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.