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Administrative data

Description of key information

In Vitro Skin Irritation (OECD 439): Non irritating.
In Vitro Eye Irritation: Irritating

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:
18 January 2012 to 09 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with current guideline and GLP compliant.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
OECD TG 439
Value:
> 50
Remarks on result:
other:
Remarks:
Exposure to Naphthoxyethanol resulted in a mean EpiSkin® viability of 91.77% ±2.70% ofthe negative control value.

MTT Direct Reduction Test (Preliminary Assay)

 

The test was scored by visual assessment of the formation of purple-coloured formazan. The positive control eugenol reduced the MTT solution to purple-coloured formazan. The negative controlwater did not reduce MTT to formazan after 3 h incubation. The test item,Naphthoxyethanol, did not reduce MTT to formazan after 3 h incubation.

 

EpiSkin® Irritation Test

Percentage Viability of EpiSkin® Tissues After ca 42h Recovery Time

Treatment

Mean viability per tissue (%)

Mean viability per test item (%)

SD (%)

2-Naphthoxyethanol

94.54

91.77

2.70

89.16

91.60

Aqueous SDS Solution

(5%, w/v)

(Positive Control)

6.20

10.52

3.91

13.83

11.54

PBS Solution

(Negative Control)

95.36

100.00

4.87

99.58

105.07

The negative control results were similar for the three viable EpiSkin® units dosed with Dulbecco’s PBS. Exposure to Dulbecco’s PBS resulted in a mean EpiSkin® viability of 100.00% ±4.87%.

 

The positive control results were similar for the three viable EpiSkin® units dosed with aqueous SDS solution (5%, w/v). Exposure to aqueous SDS solution (5%, w/v) resulted in amean EpiSkin® viability of 10.52% ±3.91%.

 

The results were similar for the three viable EpiSkin® units dosed with Naphthoxyethanol.Exposure to Naphthoxyethanol resulted in a mean EpiSkin® viability of 91.77% ±2.70% ofthe negative control value.

 

The test item was considered to be irritant to skin in accordance with GHS category 2 if the tissue viability after exposure and post-treatment incubation was less than or equal (=) to 50%.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Naphthoxyethanol was demonstrated to be non-irritant when tested in the EpiSkin® in vitro irritation assay.
Executive summary:

Evaluation of skin irritation is part of the Human Health Hazard Assessment required for registration of a chemical. In this study, the irritation potential of Naphthoxyethanol was evaluated using the SkinEthic EpiSkin in vitro irritation assay. Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan. Naphthoxyethanol did not reduce MTT to formazan. The dermal irritation potential was assessed by applying 10 mg ± 2 mg of the solid Naphthoxyethanol to the exposed surface of three EpiSkin reconstructed human epidermis (RhE) units for 15 min. Prior to applying the test item, the EpiSkin surface was pre-moistened with sterile, ultra-pure water (5 µL). The surface area of the EpiSkin was 0.38 cm2, therefore the application rate was ca 26.3 mg/cm2. After the 15 min exposure period, the test item was washed from the surface of the EpiSkin using Dulbecco’s phosphate-buffered saline (PBS). The EpiSkin was then incubated for a recovery period of ca 42 h in a humidified incubator, set to maintain temperature and CO2 levels of 37°C and 5%, respectively. Following incubation, the EpiSkin units were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for 3 h. Biopsies of the EpiSkin membranes were removed, added to acidified isopropanol, and refrigerated for ca 69 h in order to extract the

formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, aqueous sodium dodecyl sulphate (SDS) solution (5%, w/v) (10 µL), and the negative control, PBS (10 µL) were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual EpiSkin tissue was calculated as a percentage of the mean negative control viability (defined as 100%). Exposure to Naphthoxyethanol resulted in a mean EpiSkin viability of 91.77% ± 2.70% of the negative control value. Exposure to the positive control, aqueous SDS solution (5%, w/v), resulted in a mean EpiSkin viability of 10.52% ± 3.91% of the negative control value. In conclusion, Naphthoxyethanol was demonstrated to be non-irritant when tested in the EpiSkin in vitro irritation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation
Remarks:
other: In vitro assay to examine ocular irritation of a test item using the SkinEthic HCE® model.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 December 2012 - March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with current Guidelines and GLP compliant.
Qualifier:
no guideline available
Principles of method if other than guideline:
The SkinEthic HCE® model assesses the irritation potential of a test item by examining the cytotoxic effects of the test item after a defined exposure period and recovery time.

The endpoint of the HCE® assay is the estimation of cell viability by assaying the reduction of MTT by mitochondrial enzymes. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.

The SkinEthic reconstructed Human Corneal Epithelium (HCE) model has undergone ECVAM prevalidation testing and has been shown to have a high correlation with existing in vivo and in vitro data for known eye irritants and non-irritant compounds.

GLP compliance:
yes
Remarks on result:
other: Exposure to 2-Naphthoxyethanol resulted in a mean HCE viability (corrected for effect of unremoved test item) of 2.13% of the negative control value. 2-Naphthoxyethanol was demonstrated to be irritant to eyes when tested in vitro.
Irritant / corrosive response data:
Exposure to Naphthoxyethanol resulted in a mean HCE viability of 2.13% of the negative control value.

MTT Direct Reduction Test

The test was scored by visual assessment of the formation of purple-coloured formazan. The positive control (eugenol) reduced the MTT solution to purple-coloured formazan. The negative control (water) did not reduce MTT to formazan. The results of the MTT direct reduction test showed that Naphthoxyethanol did reduce MTT to formazan.

Skin Ethic HCE Irritation Test

The results of the ocular irritation assay with Naphthoxyethanol are shown in Table 1 (relative viability of the HCE tissues expressed as a percentage of the mean negative control value).

 

Table 1

Treatment

Mean Relative
Viability per
Tissue (%)

Mean Relative
Viability per
Treatment (%)

SD (%)

Naphthoxyethanol (Corrected)

2.32

2.13

0.17

2.06

2.00

Triton X-100 (0.2%, w/v)

(Positive Control)

3.48

6.54

4.01

11.08

5.06

PBS Solution

(Negative Control)

109.34

100.00

8.12

96.04

94.62

 

Negative Control

The results were similar for the three viable HCE tissues dosed with the negative control. Exposure to Dulbecco’s PBS resulted in a mean HCE viability of 100.00% ± 8.12%.

 

Positive Control

The results were similar for the three viable HCE tissues dosed with the positive control. Exposure to Triton X-100 solution (0.2%, w/v) resulted in a mean HCE viability of 6.54% ± 4.01%.

 

Naphthoxyethanol

Non-Viable Controls

The mean absorbance value of the three replicate non-viable tissues dosed with Naphthoxyethanol was 0.010 ± 0.002. The mean absorbance value of the three replicate undosed non-viable tissues was 0.045 ± 0.007. Therefore, a negative absorbance value was obtained for the effect of the unremoved test item and the calculations did not require to be adjusted.

 

Viable Tissues

The results were similar for the three viable HCE units dosed with Naphthoxyethanol. Exposure to Naphthoxyethanol resulted in a mean HCE viability of 2.13% ± 0.17% of the negative control value.

 

Histology Result

Microscopic analysis appeared to show some damage to the tissues dosed with Naphthoxyethanol. However, due to the preparation error which resulted in the lack of images for the positive control, the decision was made to reject the results of the histology and to base the conclusion of the study solely on the tissue viability results. This decision is justified, as the histology is an add-on and is not required for interpretation of irritation potential. Therefore, the integrity of the study is not compromised by the rejection of the histology data.

 

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, Naphthoxyethanol was demonstrated to be irritant to eyes when tested in vitro using the HCE® reconstructed human corneal model.
Executive summary:

Evaluation of ocular irritation is part of the Human Health Hazard Assessment required for registration of a chemical. In this study, the irritation potential of Naphthoxyethanol was assessed using the HCE in vitro ocular irritation test.

 

Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan, which is a measure of cell viability in the assay. The results of the MTT direct reduction test showed that Naphthoxyethanol did reduce MTT to formazan. Therefore, non-viable controls were tested in parallel with the irritation test to quantify this effect.

 

Eye irritation potential was assessed by applying Naphthoxyethanol (ca 30 mg) to the exposed surface of three HCE tissues for 60 min. Prior to applying the test item, the HCE surface was pre-moistened with sterile, ultra-pure water (30 µL). The surface area of the HCE was 0.5 cm2, therefore the application rate was ca 60 mg/cm2. After the 60 min exposure period, the test item was washed from the surface of the HCE using Dulbecco’s phosphate-bufferedsaline (PBS; 25 mL) and cotton swabs. Additional aliquots of PBS were required to remove all the applied Naphthoxyethanol, which had adhered to the tissue surface. The HCE tissues were then incubated for a recovery period of 16 h in a humidified incubator set to maintain temperature and CO2levels of 37°C and 5%, respectively. Following incubation, the HCE tissues were transferred to maintenance medium containing MTT (0.5 mg/mL) and returned to the incubator for 3 h. The HCE tissues were then transferred to isopropanol in order to extract the formazan. After extraction (90 min), the formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, Triton X-100 solution (0.2%, w/v), and the negative control, PBS, were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual HCE tissue was calculated as a percentage of the mean negative control viability (defined as 100%).

 

In addition to the three tissues assessed for viability, a fourth HCE tissue was dosed for each treatment as above and prepared for histological analysis. Cross-sections of each tissue were formalin-fixed, paraffin embedded and stained by the standard haematoxylin-eosin method. The tissues were then examined microscopically for tissue damage. Due to a preparation error, the results of the histological analysis were rejected as it was not possible to evaluate the positive control. Therefore, the study conclusion was based solely on the tissue viability results. The integrity of the study was unaffected as the histological evaluation isan add-on and is not required for interpretation of irritation potential. There was little visual difference between the test and negative control samples.

Exposure to Naphthoxyethanol resulted in a mean HCE viability of 2.13 ± 0.17% of the negative control value. Exposure to the positive control, Triton X-100 (0.2%, w/v), resulted in a mean HCE viability of 6.54 ± 4.01% of the negative control value. Cell viability values below a threshold of 50% of the negative control viability indicate that the test item is irritant.

 In conclusion, Naphthoxyethanol was demonstrated to be irritant to eyes when testedin vitrousing the HCE reconstructed human corneal model.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The results of the in vitro skin irritation study conducted in accordance with OECD 439 guidance and to GLP standards showed EC 202-228-8 to be non irritating. This result combined with the lack of irritation seen in the acute dermal study up to the limit dose (2000 mg/kg) is sufficient to conclude that EC 202-228-8 is non irritating to the skin. This testing strategy conforms with that outlined in REACH Annex VII and VIII.

The results from the in vitro eye irritation study conducted in accordance with current guidelines and to GLP revealed EC 202-228-8 to be irritating, as such in the interest of animal welfare it was considered unethical to proceed onto an in vivo eye irritation study. Although the guidelines are not yet validated, it is considered that the finding from the in vitro ocular test is good evidence that EC 202-228-8 is an eye irritant. Therefore the results from the in vitro assay and other relevant in vivo dermal studies will be used to determination of classification.


Justification for selection of skin irritation / corrosion endpoint:
Study conducted in accordance with OECD guidelines and GLP. Klimisch score 1.

Justification for selection of eye irritation endpoint:
Study conducted in accordance with current Guidelines and GLP compliant. Klimisch score 1.

Effects on eye irritation: irritating

Justification for classification or non-classification

The results of the in vitro skin irritation conclude EC 202-228-8 to be non irritating. There were no signs of dermal irritation in the in vivo acute dermal toxicity study up to the limit dose tested (2000 mg/kg); therefore, no classification for skin corrosion or irritation is required according to CLP.

The in vitro eye irritation study found EC 202-228-8 to be an eye irritant, it was therefore concluded in the interest of animal welfare it would be unethical to proceed to an in vivo eye irritation study. Therefore the eye irritation classification was determined using a weight of evidence approach. It is considered that the Testing And Evaluation Strategy in OECD TG 405 supports the view that there is no evidence that would require EC 202-228-8 to be classified as CLP Cat 1 (irreversible effects on the eye). It is considered that EC 202-228-8 does not have the potential to cause corrosion or serious eye damage because the in vitro skin test showed it to be non-irritating and the acute dermal toxicity test revealed no toxicity and no dermal effects after a 24 h application.  Although not yet validated, it is considered that the finding from the in vitro ocular test is good evidence that EC 202-228-8 is an eye irritant, and as such should be classified as a Cat 2 for eye irritant in accordance with CLP.