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Endpoint summary

Description of key information

Endpoint summary “irritation/corrosion”

Skin irritation: 1 in vitro studies available: not irritant;

Eye irritation: 1 studies available: not irritant

 

There is in one in vitro study available on the skin irritancy potential. Test item, Licowax R 21 S FL was applied topically to the EPISKIN epidermal model (three epidermis units were used per test item, positive and negative controls) for 15 minutes. Exposure to the test item was terminated by rinsing with Dulbecco’s phosphate buffered saline (DPBS). Epidermis was then incubated at 37°C for 42 additional hours. The viability was assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/mL; 2 mL per well). The % viability of tissue treated with the test item was 93.78% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence under the conditions of the study the test item was found to be non irritant to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemicals.

The eye damage of Licowax R 21 S FL was tested through topical application for approximately 240 minutes. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas (309.8 to 386.0 mg). The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 124 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Licowax R 21 S FL did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.3 after 240 minutes of treatment.

Since Licowax R 21 S FL induced an IVIS ≤ 3, no classification is required for eye irritation.

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:

December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Details on animal used as source of test system:

n.a.; in vitro skin model

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-TEST
The test item was tested for its ability to direct formazan reduction. This pre-test was done before starting the main study.
- 6 well of 12 well plate was filled with 2 mL of MTT solution (0.3 mg/L).
- To above 6 wells, 10 mg of the test item was added to 3 wells and 10 µL of buffer to other 3 wells and mixed well.
- Incubated the mixture for 3 hours at 37°C protected from light.
Since, no change in the color (blue or purple) of MTT solution was observed, the test item was identified to have no interaction with MTT.

MAIN STUDY
The assay medium supplied with the kits was kept at 2-8°C (refrigerator).
a) Maintenance medium was prewarmed to 37°C before using.
b) EPISKIN Standard Model™ kit was kept at room temperature in the microbiological safety cabinet until next step.

PREPARATION AND PRE INCUBATION (DAY 0)
This step was conducted in a microbiological safety cabinet
a) 3 wells of a 12 well assay plates were filled with pre-warmed maintenance medium (2 mL per well).
b) EPISKIN Standard Model™ kit was opened and epidermis units were transferred into maintenance medium filled wells, using sterile forceps.
Labelled plate lids by replicate treatment order as follows
Negative control Positive control Test item
Rep 1 (NCR1) Rep 1 (PCR1) Rep 1 (5740R1)
Rep 2 (NCR2) Rep 2 (PCR2) Rep 2 (5740R2)
Rep 3 (NCR3) Rep 3 (PCR3) Rep 3 (5740R3)
Key: Rep/R: Replicate; NC: Negative control; PC: Positive control; 5740= study number for test item Licowax R 21 S FL
c) Incubated at 37 °C, 5% CO2, in a > 95% humidified atmosphere for 24 hours.

APPLICATION OF TEST ITEM AND RINSING (DAY 1)
a) Pre-warmed maintenance medium (2 mL per well) was filled taking into account of the 3 wells per replicate. Plate lids were labeled with test-item (3 wells per test item), or negative control (3 wells) or positive control (3 wells).
Topical applications: 15 minutes treatment
b) 10 μL of negative control and positive control, 10 mg of the test item were applied on the top of the epidermis.
c) Followed the defined application order (Rep 1, Rep 2 and Rep 3).
d) After application, plate containing the treated epidermis was closed with lid and placed in the same cabinet for 15 minutes (± 0.5 minute) at room temperature.
For positive control, re-spread after 7 minutes contact time. And each tissue applications were done at 1 minute interval.
End of the treatment and removal of the test chemical; After 15 minutes exposure:
e) Treated epidermal units were removed using forceps, and rinsed thoroughly with 25 mL sterile DPBS by filling and emptying the tissue inserts to remove residual material from the epidermal surface.
f) Epidermal units were placed on an absorbent paper and remaining DPBS were removed from the epidermal surface by gently taping, and swept the epidermal surface with a cotton-bud without damaging the epidermis.
g) Blotted tissue units were transferred to the new maintenance medium pre-filled wells.
Post treatment incubation:42 hours
Incubated the treated and rinsed epidermis at 37°C, 5% CO2, 95% humidified atmosphere for 42 hours.

MTT TEST AFTER THE 42 hours INCUBATION PERIOD
Required number of wells of the assay plate were filled with 2 mL per well of 0.3 mg/mL MTT in assay medium.
Treated units were transferred to the MTT filled wells. Plate was covered with lid and incubated for 3 hours at 37°C, 5% CO2, 95% humidified atmosphere.

FORMAZAN EXTRACTION (DAY 3)
a) 1.5 mL of conic “safelock” type micro tubes were labelled appropriately for test item, vehicle control, positive control and their replicate Number (1 tube/epidermis).
Formazan extraction :
b) Tissue units were placed on absorbent paper to dry the tissues.
c) Using the special biopsy punch, a total biopsy of the epidermis was done.
d) Gently separated the epidermis from the collagen matrix with the aid of forceps, and both parts (epidermis and collagen matrix) were placed into the labelled micro tubes.
e) When all the tissues were punched and ready in the micro tubes, 500 μL of acidic isopropanol per tube was added.
f) Each tube was plugged to avoid evaporation and mixed thoroughly using a vortex mixer.
g) All the tissues were correctly immersed in the solvent and extraction start time for each tissue was noted.
h) The tubes were stored for 4 hours at room temperature (recommended 18 to 23°C) and protected from light.
i) Vortex each tube at the middle of the incubation period.
j) Tissues were removed from each tube extraction end time was noted.
k) Extraction solution was homogenized by pipetting 3 times up and down.
l) From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in using spectrophotometer at 560 nm. Acidified isopropanol solution was used as blank.

Control samples:

other: 5% SDS, DPBS

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

93.78

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The negative controls mean OD was found to be 0.726, which is well within the acceptability range. The % viability of tissue treated with the positive control was 24.13% and it was less than 50% of the negative control, which reflects the irritant nature of positive control and the sensitivity of the tissues used in the study.

Interpretation of results:

GHS criteria not met

Conclusions:

The % viability of tissue treated with the test item was 93.78% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence under the conditions of the study the test item was found to be Non Irritant to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemicals.

Executive summary:

Test item, Licowax R 21 S FL was applied topically to the EPISKIN^TMepidermal model (three epidermis units were used per test item, positive and negative controls) for 15 minutes. Exposure to the test item was terminated by rinsing with Dulbecco’s phosphate buffered saline (DPBS). Epidermis was then incubated at 37°C for 42 additional hours. The viability was assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/mL; 2 mL per well). The precipitated formazan was then extracted using acidified isopropanol (0.5 mL) for 4 hours at room temperature, and quantified spectrophotometrically at 560 nm using 96 well plates (200 μL/well).

 

SDS 5% and DPBS treated epidermis were used as positive (PC) and negative controls (NC) respectively. For each treated tissue the viability was expressed as the % of the negative control tissues (mean).

 

The negative controls mean OD was found to be 0.726, which is well within the acceptability range. The % viability of tissue treated with the positive control was 24.13% and it was less than 50% of the negative control, which reflects the irritant nature of positive control and the sensitivity of the tissues used in the study.

 

The % viability of tissue treated with the test item was 93.78% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence under the conditions of the study the test item was found to beNonIrritantto skin in accordance with UN GHSas specified in the OECD Guideline for the Testing of Chemicals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2015 - 15 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Species:

cattle

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Since no workable suspension of the test item could be obtained, it was added pure on top of the corneas.
- Amount applied: 309.8 to 386.0 mg per cornea, ensuring complete coverage

NEGATIVE CONTROL
- Amount applied: 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µl 20% (w/v) Imidazole solution

Duration of treatment / exposure:

240 (+/- 10 minutes)

Number of animals or in vitro replicates:

Test item: 3
Negative control: 3
Positive control: 3

Details on study design:

TEST SITE
- Isolated bovine cornea
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

REMOVAL OF TEST SUBSTANCE
- Washing: yes, at least three times with MEM (with phenol red)
- Time after start of exposure: 240 minutes
- Determination of permeability: After exposure the corneas (anterior compartments) were incubated in sodium-fluorescein (5 mg Na-fluorescein/ml cMEM solution) for 90 ± 5 minutes (both incubations at 32 ± 1°C). The optical density at 490 nm (OD490) was measured in triplicate using a microplate reader.

SCORING SYSTEM:
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

TOOL USED TO ASSESS SCORE:
- opacitymeter (OP-KIT) and microplate reader (TECAN Infinite® M200 Pro Plate Reader)

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritant (UN GHS: catgegory 1);
For a test substance that induces an IVIS >3 and ≥ 55, no prediction on irritant potency can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

-1.3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- The individual in vitro irritancy scores for the negative controls ranged from -2.7 to -0.4. The individual positive control in vitro irritancy scores ranged from 96 to 149. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
- The corneas treated with test item showed opacity values ranging from -2.1 to -1.2 and permeability values ranging from 0.007 to 0.032. The corneas were clear after the 240 minutes of treatment. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.0 to -0.7 after 240 minutes of treatment.
- Negative and positive control responses were within the historical data range, therefore the acceptability criteria were met and the system was considered to function properly.

Interpretation of results:

GHS criteria not met

Remarks:

(also not classified according to CLP criteria)

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, it is concluded that Licowax R 21 S FL is not irritant or corrosive for the eye under the experimental conditions described in this report. Therefore, no classification is required according to GHS and CLP criteria.

Executive summary:

The eye damage of Licowax R 21 S FL was tested through topical application for approximately 240 minutes.

Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas (309.8 to 386.0 mg).

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 124 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Licowax R 21 S FL did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.3 after 240 minutes of treatment.

Since Licowax R 21 S FL induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

With reference to the results of the in vitro skin irritation study the registration substance does not have to be classified as irritant to the skin according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

Licowax R 21 S FL induced an IVIS ≤ 3, no classification is required for eye irritation.

Therefore it is concluded that the undiluted registration substance does not have to be classified as irritant to the eyes according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).