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Administrative data

Description of key information

3 studies were performed on the analogue substance:
- an in vitro skin irritation test showed no irritation
- an in vitro eye irritation test showed no irritation or corrosion
- an in vivo rabbit eye irritation test showed no irritation or corrosion

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:
Start : 11 March 2013 Completion : 19 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deficiencies
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)
Species:
other: adult human-derived epidermal keratinocytes
Details on test animals or test system and environmental conditions:
See details on cell culture in Details on Study Design section
Vehicle:
unchanged (no vehicle)
Details on study design:
EPISKIN Small Model (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-009) was used in the study. 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.

On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France. All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.9 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 25 μl of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance

The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell viability measurement

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Irritation / corrosion parameter:
other: other: cell viability
Value:
ca. 102
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Reversibility: other: no adverse effect therefore reversibility is not relevant. Remarks: The test substance showed 102% cell viability compared to controls and was therefore not irritating.

Alkylated Naphthalene was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that Alkylated Naphthalene did not interact with MTT.

The mean absorption at 570 nm measured after treatment with Alkylated Naphthalene and controls are presented below:

Negative control      1.069  ±  0.113

Test Substance        1.094  ±  0.032

Positve control         0.207  ±   0.013

The mean tissue viability obtained after 15 minutes treatment with Alkylated Naphthalene compared to the negative control tissues is shown below:

Negative control       100 %

Test substance       102 %

Positive control       19  %

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Since the mean relative tissue viability for FAlkylated Naphthalene was above 50%, Alkylated Naphthalene is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 19%. The absolute mean OD570 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 13%, indicating that the test system functioned properly.

Interpretation of results:
GHS criteria not met
Conclusions:
Alkylated Naphthalene is non-irritant in the in vitro skin irritation test.
Executive summary:

An in vitro skin irritation test was conducted with Alkylated Naphthalene using a human skin model.

This study evaluates the ability of Alkylated Naphthalene to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of Alkylated Naphthalene was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 18515 of Alkylated Naphthalene was a clear amber slightly viscous liquid with a purity of UVCB (100%). Alkylated Naphthalene was applied undiluted (25 μl directly on top of the skin tissue for 15 minutes. After a 2 hour post-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 MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

The relative mean tissue viability obtained after 15 minutes treatment with Alkylated Naphthalene compared to the negative control tissues was 102%. Since the mean relative tissue viability for Alkylated Naphthalene was above 50% after 15 minutes treatment Alkylated Naphthalene is considered to be non-irritant.

The positive control had a mean cell viability of 19% after 15 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 13%, indicating that the test system functioned properly.

It is concluded that this test is valid and that Alkylated Naphthalene is non-irritant in the in vitro skin irritation test under the experimental conditions of the study.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study in-life phase Start : 15 April 2013 Completion : 25 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study without deficiencies
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Species: Albino rabbit, New Zealand White, (SPF-Quality).

Source: Charles River France, L’Arbresle Cedex, France
Number of animals 3 Males.
Age and body weight Animals used within the study were between 12 and 24 weeks old and
body weights were at least 1.5 kg.
Identification Earmark.
Health inspection At least prior to dosing. It was ensured that the animals were healthy and
that the eyes were free from any abnormality.

Animal husbandry
Conditions:
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40
to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any
variations to these conditions were maintained in the raw data and had no effect on the outcome of the
study.
Accommodation
Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany,
dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
Acclimatization period was at least 5 days before start of treatment under laboratory conditions.
Diet
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy)
approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden
sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study
period
Water
Free access to tap water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed
according to facility standard procedures. There were no findings that could interfere with the study.
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
3
Details on study design:
Each animal was treated by instillation of 0.1 mL of the test substance, in the conjunctival sac of one
of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held
together for about one second to prevent loss of the test substance. The other eye remained untreated
and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany)
in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine
corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated
as a percentage of the total corneal area.
In order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam
0.5 mg/kg were administered by subcutaneous injection following the 24 hours observation.
After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%
(AST Farma BV, Oudewater, The Netherlands).

Observations:
Mortality/Viability Twice daily.
Toxicity At least once daily.
Body Weight Day of treatment (prior to instillation) and after the final observation.
Necropsy No necropsy was performed according to protocol.
Irritation The eyes of each animal were examined approximately 1, 24, 48 and 72
hours after instillation of the test substance. The irritation scores and a
description of all other (local) effects were recorded.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 24
Score:
ca. 0
Max. score:
0
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 48
Score:
ca. 0
Max. score:
0
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 72
Score:
ca. 0
Max. score:
0
Irritant / corrosive response data:
Instillation of 0.1 mL of Alkylated Naphthalene into one eye of each of three rabbits resulted in irritation of the conjunctivae,
which consisted of redness, chemosis and discharge. The irritation had completely resolved within 24
hours.
No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24
hours after test substance instillation revealed no corneal epithelial damage.

There was no evidence of ocular corrosion.
Other effects:
No mortality or clincal symptoms
Interpretation of results:
GHS criteria not met
Conclusions:
Alkylated Naphthalene is not irritating or corrosive to the eye.
Executive summary:

An acute eye irritation/corrosion study was conducted with Alkylated Naphthalene in the rabbit. The study was carried out based on the guidelines described in: OECD No.405 (2012) "Acute Eye Irritation / Corrosion". Single samples of 0.1 mL of Alkylated Naphthalene were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation. Instillation of the test substance resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 24 hours. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. There was no evidence of ocular corrosion. Based on these results, Alkylated Naphthalene does not have to be classified and has no obligatory labeling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures including all amendments.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start : 12 March, 2013 Completion : 12 March, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deficiencies
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Bovine eyes
Details on test animals or tissues and environmental conditions:
See Study Design Section
Vehicle:
unchanged (no vehicle)
Details on study design:
Study design

Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation were discarded.
The isolated corneas were stored at 32 ± 1°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) 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.
Cornea selection and 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 (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck, Darmstadt, Germany) 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 4 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.
Permeability determinations
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 (TECAN Infinite® M200 Pro Plate Reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.
Irritation parameter:
in vitro irritation score
Run / experiment:
OECD 437
Value:
ca. 1.094
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Treatment

Mean

Opacity1

Mean

Permeability1

Mean In vitro Irritation Score1, 2

Negative control

0

0.000

0.0

Positive control

(Benzalkonium Chloride)

75

3.174

123

Alkylated Naphthalene

2

-0.057

1.1

 

1 Calculated using the negative control mean opacity and mean permeability values.

2In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

Mean tissue viability in thein vitroskin irritation test with Alkylated Naphthalene Mean tissue viability (percentage of control)

Negative control

100

Test substance

102

Positive control

19

Interpretation of results:
GHS criteria not met
Conclusions:
Alkylated Naphthalene is not a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test.
Executive summary:

Screening for the eye irritancy potential of Alkylated Naphthalene was conducted using the Bovine Corneal Opacity and Permeability test (BCOP test). This test describes the ocular irritation properties of Alkylated Naphthalene on an isolated bovine cornea. The ocular irritancy of the test substance was tested through topical application for approximately 10 minutes. The study procedures were based on the most recent OECD and EC guideline.

The test substance was applied neat (750 μl) directly on top of the corneas. 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, except the permeability of one of the corneas. However since the permeability scores of the other two eyes were within the historical control data, the validity of the test was considered to be not affected. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 123 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Alkylated Naphthalene did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment. Finally, it is concluded that this test is valid and that Alkylated Naphthalene is not a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions of the test.

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

Additional information

The analogue alkylated naphthalene was shown in all tests to lack irritation or corrosion properties for eye and skin.

Justification for classification or non-classification

Based on lack of irritation in in vitro and in vivo testing on the analogue, the substance does not have to be classified for skin or eye irritation according to Regulation (EC) No 1272/2008.