Amides, C8-10, N-(hydroxyethyl)

Endpoint summary

Description of key information

There is evidence for a skin irritating potential of Amides, C8-10, N-(hydroxyethyl) from an in-vitro skin irritation study (OECD guideline 439), therefore Amides, C8-10, N-(hydroxyethyl) is classified for skin irritation category 2 / R38, according to CLP (EU-GHS) Regulation (EC) No1272/2008 and Directive 67/548/EEC, respectively. 
Corrosive effects on the bovine cornea were observed in an in-vitro BCOP study according to OECD guideline 437, therefore Amides, C8-10, N-(hydroxyethyl) is classified for eye irritation category 1 / R41, according to CLP (EU-GHS) Regulation (EC) No1272/2008 and Directive 67/548/EEC, respectively. 

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:

2012-04-25 to 2012-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In Vitro Skin Irritation: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human epidermis model EpiDerm™ (EPI-200-KIT)

Details on test animals or test system and environmental conditions:

The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Amount / concentration applied:

Approximately 11 to 13 mg of fine ground test material was applied to the tissues and wetted each with 15 μL deionised water. It was taken care that the test item was evenly distributed on the tissues. For the positive and negative controls 10 μL were dosed per tissue.

Duration of treatment / exposure:

Positive control 15 min (± 1 min) (5% SLS)
Negative control 15 min (± 1 min) Deionised water
Test substance 15 min (± 1 min)

Observation period:

after washing process post incubation period of 42 h

Number of animals:

The test was performed in triplicate

Irritant / corrosive response data:

Test substance: mean viability 5.3 (+/- 0.7) %
Negative control: mean viability 100 (+/- 10.5) %
Positive control: mean viability 20.8 (+/- 1.0) %

Test substance: mean viability 5.3 (+/- 0.7) %

Negative control: mean viability 100 (+/- 10.5) %

Positive control: mean viability 20.8 (+/- 1.0) %

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Topical application of the test substance on top of the stratum corneum of the reconstructed human epidermis model EpiDerm™ (in triplicate) resulted in a mean cell viability of 5.3 % when compared to the corresponding negative control. According to the evaluation scheme of OECD test guideline 439 the test substance has to be predicted as irritating to the skin.

Executive summary:

The skin irritating potential of the test substance Amides, C8-10, N-(hydroxyethyl) has been investigated using an in-vitro reconstructed human epidermal model (RHE), according to OECD guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, July 2010).

EpiDerm™ RHEs (EPI-200-KIT) were topically exposed to 11 to 13 mg of fine ground test material for 15 min. The cell viability was determined after an additional 42 h (± 2 h) post incubation period. For the analysis of the tissue viability the Standard MTT Assay was used. The viability of the negative control (Deionised water) was set to 100%.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 5.3% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The sensitivity and accuracy of the used test system has been demonstrated with the reference substance SLS (5% aqueous solution). A mean cell viability of 20.8 % in relation to the corresponding negative control Deionised water was determined.

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance Amides, C8-10, N-(hydroxyethyl) is irritant to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2012-04-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

December 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted September 07, 2009

Deviations:

no

GLP compliance:

yes

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring)
of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

Vehicle:

physiological saline

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water)

Duration of treatment / exposure:

240±10 minutes

Number of animals or in vitro replicates:

triplicates

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes; at least 3 times with saline
- Time after start of exposure: 240±10 min

SCORING SYSTEM: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value); a test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

TOOL USED TO ASSESS SCORE: opacitometer; permeability measurement with sodium fluorescein

Negative control: physiological saline
Positive control: 10% (w/v) Benzalkonium chloride in saline

Irritant / corrosive response data:

The individual in vitro irritancy scores for the negative controls ranged from 2.62 to 3.1 for the corneas. The individual positive control in vitro irritancy scores ranged from 178.35 to 264.03. The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity effects on the corneae (mean in vitro irritation score 217.33) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).
The corneas treated with the test substance showed opacity values ranging from 8.33 to 16.33 and permeability values ranging from 0.902 to 1.957. The in vitro irritancy scores ranged from 21.86 to 45.69 after 240 minutes of treatment with the test substance.

Treatment	Mean Opacity	Mean Permeability	MeanIn vitro Irritation Score
Negative control	1.67	0.078	2.84
Positive control	217.33	0	217.33
Test substance	11.66	1.482	33.91

Relative to the negative control, the test item Amides, C8-10, N-(hydroxyethyl) caused an increase of the corneal opacity and strong permeability effects occurred. The calculated mean in vitro irritation score was 33.91. According to OECD 437 the test item could be classified as not

corrosive / not severe irritant to the eye. But the peeling of the upper cell layers of the corneae exposed to the test item when rinsing after the treatment period contradicts classifying the test item as non corrosive / non severe irritant.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.84). The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity effects on the corneae (mean in vitro irritation score 217.33) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Interpretation of results:

other: not corrosive to the eye (CLP/EPA/GHS (Cat 1)), if considering the results of the opacity and permeability measurement after exposure to the corneae, but the lost of the upper cell layers indicates a corrosive effect.

Remarks:

Criteria used for interpretation of results: other: CLP, EU GHS (Regulation (EC) No 1272/2008)

Conclusions:

Amides, C8-10, N-(hydroxyethyl) is considered to be not corrosive to the eye (CLP/EPA/GHS (Cat 1)), if considering the results of the opacity and permeability measurement after exposure to the corneae, but the lost of the upper cell layers indicates a corrosive effect.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Amides, C8-10, N-(hydroxyethyl) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 7 September 2009 and EU method B.47, December 2010.

The test substance was tested as 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water). Physiological saline was used as negative control, 10% (w/v) Benzalkonium chloride in saline as positive control.

The corneas were incubated with Amides, C8-10, N-(hydroxyethyl) solution f

or 240±10 min at 32±1°C. The test was performed in triplicates. After rinsing the corneas at least 3 times, opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

The positive and negative controls induced the appropriate responses.

The corneas treated with Amides, C8-10, N-(hydroxyethyl) showed opacity values ranging from 8.33 to 16.33 and permeability values ranging from 0.902 to 1.957. The in vitro irritancy scores ranged from 16 to 44. The mean in vitro irritancy score was 33.91 after 240 minutes of treatment with Amides, C8-10, N-(hydroxyethyl). But the peeling of the upper cell layers of the corneae exposed to the test item when rinsing after the treatment period period contradicts classifying the test item as non corrosive / non severe irritant.

In conclusion, according to the current study and under the experimental conditions reported,

the test item Amides, C8-10, N-(hydroxyethyl) is not corrosive to the eye (CLP/EPA/GHS (Cat 1)), if considering

the results of the opacity and permeability measurement after exposure to the corneae, but

the lost of the upper cell layers indicates a corrosive effect.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The skin irritating potential of the test substance hexanoic acid, therefore Amides, C8-10, N-(hydroxyethyl) has been investigated using an in-vitro reconstructed human epidermal model (RHE), according to OECD guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, July 2010).

EpiDerm™ RHEs were topically exposed to 30µL of the test substance for 60 min (± 1 min). The cell viability was determined after an additional 42 h (± 2 h) post incubation period. For the analysis of the tissue viability the Standard MTT Assay was used. The viability of the negative control (UPW) was set to 100%.

The topical application of the test substance on top of stratum corneum of the RHE (each in triplicate) resulted in a mean cell viability of 5.3% related to the corresponding negative control.

Residual test substance adhered to the reconstructed epidermis after completion of the rinsing procedure may have had an impact on the overall test performance due to the chemically reducing properties of the test substance towards the MTT reagent. A control with freeze killed tissue, to identify possible interference, was not included in this study. However, according to the study authors the validity of the study was not affected.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The sensitivity and accuracy of the used test system has been demonstrated with the reference substance SLS (5% aqueous solution). A mean cell viability of 20.8 % in relation to the corresponding negative control Deionised water was determined.

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, the testsubstance Amides, C8-10, N-(hydroxyethyl) is irritant to skin.

Eye irritation

Data from an in-vitro Bovine Corneal Opacity and Permeability (BCOP) test, an accepted alternative method to the Draize test, to evaluate the acute irritation potential are available.

The test substance was tested as 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water). Physiological saline was used as negative control, 10% (w/v) Benzalkonium chloride in saline as positive control.

The positive and negative controls induced the appropriate responses.

The corneas treated with Amides, C8-10, N-(hydroxyethyl)showed opacity values ranging from 8.33 to 16.33 and permeability values ranging from 0.902 to 1.957. The in vitro irritancy scores ranged from 16 to 44. The mean in vitro irritancy score was 33.91 after 240 minutes of treatment with Amides, C8-10, N-(hydroxyethyl).But the peeling of the upper cell layers of the corneae exposed to the test item when rinsing after the treatment period period contradicts classifying the test item as non corrosive / non severe irritant.

In conclusion, according to the current study and under the experimental conditions reported,

the test item Amides, C8-10, N-(hydroxyethyl) is not corrosive to the eye (CLP/EPA/GHS (Cat 1)), if considering

the results of the opacity and permeability measurement after exposure to the corneae, but

the lost of the upper cell layers indicates a corrosive effect.

In conclusion the test substance has to be evaluated as severely irritating including irreversible effects on the eye.

Justification for selection of skin irritation / corrosion endpoint:
Data from a GLP compliant in-vitro guideline study with reliability 1.

Justification for selection of eye irritation endpoint:
Data from a GLP compliant guideline study with reliability 1.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

Based on the available in-vitro skin irritation study, the key study demonstrated that an exposure of reconstructed human epidermis to

amides, C8-C10, N-(hydroxyethyl) clearly indicated irritation to skin. Therefore based on the

data derived from the key study, R38 classification according to EC criteria (67/548/EEC) and skin irritation

category 2; H315-causes skin irritation (hazard statement) according to CLP criteria (EC 1272/2008) are

warranted.

 

Classification of eye irritation is based on in-vitro data from a Bovine Corneal Opacity and Permeability (BCOP) test according to OECD Guideline 437. The lost of the upper cell layers indicates a corrosive effect.

In conclusion the test substance has to be evaluated as severely irritating including irreversible effects on the eye.