2-octyldodecyl 5-oxo-L-prolinate

Endpoint summary

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:

From August 22, 2016 to August 29, 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

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch no.: CH 192955/01
Purity/composition: 99.98%
Appearance: clear yellow liquid

Test system:

human skin model

Remarks:

human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM))

Cell type:

non-transformed keratinocytes

Justification for test system used:

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 was 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. 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:

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:

25 µL

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42h incubation period
(After the 42 hour 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 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

MTT test

Run / experiment:

skin irritation test on human three dimensional epidermal models

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Skin irritation was expressed as the remaining cell viability after exposure to the test substance.
- The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test substance was above 50%, it was considered to be non-irritant.
- The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.
The standard deviation value of the percentage viability of three tissues treated identically was less than 7%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Executive summary:

A study was conducted to determine the in vitro skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46 (Reconstructed Human Epidermis Test Method), in compliance with GLP. Human three dimensional epidermal models (triplicates) were exposed to 25µL undiluted test substance for 15 min. After a 42 hour post-incubation period, a determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The positive control (25 µL 0.5% SDS) had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control (25 µL PBS) tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 97%. The standard deviation value of the percentage viability of three tissues treated identically was less than 7%, indicating that the test system functioned properly. Th experiment was considered valid. Since the mean relative tissue viability for the test substance was above 50% after 15 ± 0.5 minutes treatment, the test substance was considered to be non-irritant to human skin under the study conditions (Eurlings, 2016).

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

Remarks:

Bovine Corneal Opacity and Permeability(BCOP) test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 09, 2016

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:

yes

Remarks:

but the study integrity was not adversely affected by the deviation

GLP compliance:

yes

Specific details on test material used for the study:

Batch no.: CH 192955/01
Purity/composition: 99.98%
Appearance: clear yellow liquid

Species:

cattle

Details on test animals or tissues and environmental conditions:

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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

other: positive control: ethanol (purity: > or = to 99.9%).

Amount / concentration applied:

750 µL of undiluted test substance

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours (followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein)

Number of animals or in vitro replicates:

3

Details on study design:

The Bovine Corneal Opacity and Permeability (BCOP) test is an organic model that provides short-term maintenance of normal physiological and biological function of the bovine cornea in an isolated system. In this test method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitymeter and an ultraviolet/visible spectrophotometer, respectively.
1. Preparation of corneas
The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.
2. Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded.
3. Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C. After the incubation the solutions and the test substance were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
4. Opacity measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = (I0/I - 0.9894)/0.0251 (with I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea).
5. Application of sodium fluorescein and Permeability determinations
Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1°C. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader.

Irritation parameter:

in vitro irritation score

Run / experiment:

in vitro irritancy score (opacity and permeability)

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- The individual in vitro irritancy scores for the negative controls ranged from 0.5 to 1.5. 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 individual positive control in vitro irritancy scores ranged from 53 to 72 for ethanol. The corneas treated with the positive control substance were translucent after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control was 62 and was within two standard deviations of the current historical positive control mean.
Based on the results, it was therefore concluded that the test conditions were adequate and that the test system functioned properly.
- The corneas treated with the test substance showed opacity values ranging from 0.0 to 1.5 and permeability values ranging from -0.032 to -0.016. The corneas were clear after the 10 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.2 to 1.0 after 10 minutes of treatment with the test substance. The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment. Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Interpretation of the results:

- In vitro irritancy score

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).

Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

< or = 3: no category,

>3 and < or = to 55: no prediction can be made,

> 55: category 1.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance did not induce ocular irritation through both endpoints (cornea opacity and permeability) and was therefore considered non-irritant to cattle eye.

Executive summary:

A study was conducted to determine the in vitro eye irritation potential of the test substance according to OECD guideline 437 (bovine corneal opacity and permeability (BCOP) test), in compliance with GLP. Bovine corneas were exposed to the undiluted test substance for a period of 10 minutes (followed by a post-incubation of 2h with fresh medium). Opacity and permeability were measured and an in vitro irritancy score was then established. Physiological saline and ethanol were used as negative and positive controls, respectively. The corneas treated with the test substance showed opacity values ranging from 0.0 to 1.5 and permeability values ranging from -0.032 to -0.016. The corneas were clear after the 10 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. The in vitro irritancy scores ranged from -0.2 to 1.0 after 10 minutes of treatment. The individual in vitro irritancy scores ranged from 0.5 to 1.5 for the negative control and from 53 to 72 for the positive control. The corneas treated with the positive control were transluscent after the 10 minutes of treatment. The negative and positive control values were within the expected range; hence the experiment was considered valid. Under the study conditions, the test substance did not induce ocular irritation through both endpoints to cattle eye, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment. Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage (Eurlings, 2016).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation study:

A study was conducted to determine the in vitro skin irritation potential of the test substance according to OECD Guideline 439 and EU Method B.46 (Reconstructed Human Epidermis Test Method), in compliance with GLP. Human three dimensional epidermal models (triplicates) were exposed to 25µL undiluted test substance for 15 min. After a 42 hour post-incubation period, a determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The positive control (25 µL 0.5% SDS) had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control (25 µL PBS) tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 97%. The standard deviation value of the percentage viability of three tissues treated identically was less than 7%, indicating that the test system functioned properly. Th experiment was considered valid. Since the mean relative tissue viability for the test substance was above 50% after 15 ± 0.5 minutes treatment, the test substance was considered to be non-irritant to human skin under the study conditions (Eurlings, 2016).

In vitro/ex vivo eye irritation study:

A study was conducted to determine the in vitro eye irritation potential of the test substance according to OECD guideline 437 (bovine corneal opacity and permeability (BCOP) test), in compliance with GLP. Bovine corneas were exposed to the undiluted test substance for a period of 10 minutes (followed by a post-incubation of 2h with fresh medium). Opacity and permeability were measured and an in vitro irritancy score was then established. Physiological saline and ethanol were used as negative and positive controls, respectively. The corneas treated with the test substance showed opacity values ranging from 0.0 to 1.5 and permeability values ranging from -0.032 to -0.016. The corneas were clear after the 10 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. The in vitro irritancy scores ranged from -0.2 to 1.0 after 10 minutes of treatment. The individual in vitro irritancy scores ranged from 0.5 to 1.5 for the negative control and from 53 to 72 for the positive control. The corneas treated with the positive control were transluscent after the 10 minutes of treatment. The negative and positive control values were within the expected range; hence the experiment was considered valid. Under the study conditions, the test substance did not induce ocular irritation through both endpoints to cattle eye, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment (Eurlings, 2016).

Justification for classification or non-classification

Based on the results of in vitro skin and eye irritation studies, the substance does not warrant classification for these endpoints according to CLP (EU 1272/2008) criteria.