Registration Dossier

Administrative data

Description of key information

5 studies were performed:
- an in vitro skin irritation test showed no irritation with analog Alkylated Naphthalene
- an in vivo skin irritation study obtained in the inquiry process for a substance with the same EC number as Alkylnaphthalene
- an in vitro eye irritation test showed no irritation or corrosion
- two in vivo rabbit eye irritation test showed the materials should not be classified as eye irritations

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Start : 11 March 2013 Completion : 19 March 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deficiencies. The study was conducted on a close structural analog of alkylnaphthalene.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: adult human-derived epidermal keratinocytes
Details on test animals 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:
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. (migrated information)
Irritant / corrosive response data:
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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: EPISKIN Small Model
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
Reference
Endpoint:
eye irritation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Study in-life phase Start : 15 April 2013 Completion : 25 April 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP Guideline study without deficiencies. Alkylated Naphthalene, EC Number 700-826-8 is a close structural analog of alkyl naphthalene. The substances have similar chemical and physical properites and the irritation studies for Alkylated Naphthalene, EC Number 700-826-8 can be used as read-across to alkyl naphthalene.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
8
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 48
Score:
ca. 0
Max. score:
8
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 72
Score:
ca. 0
Max. score:
8
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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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 conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

The analog Alkylated Naphthalene was shown in all tests to lack irritation or corrosion properties for eye and skin.

Test data for a substance with the same EC number (410-190-0) as alkylnaphathalene, identified during Inquiry Process, was not a skin irritant and was not an eye irritant.

An in vivo skin irritation test is not required for Alkylated Naphthalene. The substance has been investigated in an EPISKIN OECD 439 respectively Method B.46 of Commission Regulation (EC) No. 440/2008/EC and was proven to be non-irritant in this test. The EU Commission has acknowledged in its Statement on the validity of in-vitro tests for skin irritation at its 26th meeting, held on 26-27th of April 2007 for the Validation of Alternative Methods (ECVAM) unanimously that the EPISKIN method showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction, and for being used as a replacement for the in-vivo Draize Skin Irritation Test . Consequently, for animal welfare an in-vivo test is not scientifically justifiable. Furthermore, the availability of read-across data from a substance with the same EC number of alkylnaphthalene, though a full study report is not available, support the conclusion that the substance is not a skin irritant and that further testing is unnecessary.


Justification for selection of skin irritation / corrosion endpoint:
Alkylated Naphthalene has been investigated in an EPISKIN OECD 439 respectively Method B.46 of Commission Regulation (EC) No. 440/2008/EC and was proven to be non-irritant in this test. The EU Commission has acknowledged in its Statement on the validity of in-vitro tests for skin irritation at its 26th meeting, held on 26-27th of April 2007 for the Validation of Alternative Methods (ECVAM) unanimously that the EPISKIN method showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction, and for being used as a replacement for the in-vivo Draize Skin Irritation Test . Consequently, for animal welfare an in-vivo test is not scientifically justifiable. It is also relevant that the in vivo eye test showed that the substance did not require classification for irritation effects.

Justification for selection of eye irritation endpoint:
GLP in vivo eye irritation test showed that Alkylated Naphthalene is not irritating and does not need to be classified.

Justification for classification or non-classification

Based on lack of irritation in in vitro and in vivo testing, Alkylated Naphthalene does not have to be classified and has no obligatory labeling requirement for skin or 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.