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Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tis-sue surface.
One plate (3 tissues) was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tis-sue surface.
One plate (3 tissues) was used for treatment with the test item:
The tissues were wetted with 25 µL DPBS buffer before applying the test item and spread-ing it to match the tissue size.
The following amounts were applied to the tissues:

Table 7.3 a Amounts of Test Item

Tissue Amount
1 25.2 mg
2 25.2 mg
3 25.8 mg

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% rel-ative humidity. The plates were then left to stand at room temperature until the expiration of one hour (see deviation in chapter 11).
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The sur-face of the inserts was then carefully dried with a sterile cotton tipped swab.
Then, the tissues were set in the incubator for 25 hours at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Average of Tissues 1-3

Value:

97.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

101.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

95.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

97.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

After the treatment with the test item, the mean value of relative tissue viability was reduced to 97.9%. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

Therefore, the test item Stearinsäureanhydrid is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDerm^TM were treated with the test item for 60 minutes (see deviation in chapter 11).

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.537.

The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 5.4% (required: £ 20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Vehicle:

water

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

26.2 - 26.7 mg

Duration of treatment / exposure:

3 min and 1 h incubation time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean, 3 min incubation time

Value:

95.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean, 1 h incubation time

Value:

103.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test item Stearinsäureanhydrid is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test method.

Executive summary:

One valid experiment was performed.

The test item is considered non-corrosive to skin.

After 3 minutes treatment, the mean value of relative tissue viability of the test item was decreased to 95.3%. This value is above the threshold for corrosivity (50%). After 1 hour treatment the mean value of relative tissue viability of the test item was increased to 103.1%. This value is above the threshold for corrosivity (15%).

 

The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues.

The positive control has met the validity criterion too, thus ensuring the validity of the test system.

 

For these reasons, the result of the test is considered valid.

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

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 30 minutes. After that, 50 µL of the controls and a defined amount of the test item (see Table 7.2 a) were applied in duplicate in one- minute- intervals.
At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidi-ty.
After the post-treatment incubation, the MTT assay was performed.

The following amounts were applied to the tissues:

Table 7.2 a Exact amounts of Test Item

Replicate Amount
Tissue 1 50.5 mg
Tissue 2 49.9 mg

Irritation parameter:

percent tissue viability 

Run / experiment:

average of tissues 1+2

Value:

116.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 2

Value:

119.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 1

Value:

113.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). In this case no prediction can be made and further testing with other suitable test methods is re-quired.

Under the conditions of the test, Stearinsäureanhydrid is considered non eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

One valid experiment was performed.

The test item Stearinsäureanhydrid was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

The solid test item was applied to two tissue replicates.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control and methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 1.4. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 34.5% (< 50%).

The variation within tissue replicates of the controls and the test item was acceptable (< 20%).

 

After treatment with the test item, the mean value of relative tissue viability was 116.8%.

This value is above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine corneas were used. They were collected from slaughtered cattle that were be-tween 12 and 60 months old

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item is not sufficiently soluble in a concentration of 20% in olive oil or HBSS.
Therefore, the test item was tested as suspension with a concentration of 20% in olive oil.

Duration of treatment / exposure:

4 hours at 32 ± 1 °C

Number of animals or in vitro replicates:

3

Irritation parameter:

cornea opacity score

Run / experiment:

Mean Opacity Difference corrected

Value:

2.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this test, the test item Stearinsäureanhydrid showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.67.
According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Executive summary:

One valid experiment was performed.

Under the conditions of this test, the test item Stearinsäureanhydrid showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.67.

According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

 The test item was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria. The solvent control olive oil showed no adverse effects on the test system.

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available