Registration Dossier

Diss Factsheets

Administrative data

Description of key information

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:
19 August 2011 - 14 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
other: EU Method B.46 (In vitro dermal irritation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 439 (In vitro dermal irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiskinTM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C
Dates of experimental phase: from 13 September 2011 to 14 November 2011
Amount/concentration applied:
10 mg +/- 2 mg
Duration of treatment / exposure:
15 (+/- 1) minutes exposure period and a 42-hour recovery period, followed by rinsing.
Duration of post-treatment incubation (if applicable):
MTT-loading after a 42 h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells.
Number of replicates:
Not applicable.
Triplicate tissues for each timepoint and tested substance (test item, negative control, positive control)
Species:
other: reconstructed human epidermis
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
REMOVAL OF TEST SUBSTANCE
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Excess D-PBS was removed by blotting the bottom of the tissue culture insert with absorbent paper. If necessary the epidermal surface was gently swept with a cotton-bud to remove excess D-PBS (without damaging the epidermis). If the test material was not removed, this was noted in the study file. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at 37°C, 5% CO2 in a humidified incubator for 42 (± 1) hours.

POSITIVE CONTROL
Name: Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution.

NEGATIVE CONTROL
Name: Phosphate-Buffered Saline (PBS).

SCORING SYSTEM:
- Optical density (OD) was measured between 540 and 595 nm.
Relative mean viability (%) = 100 x mean OD(test item) / mean OD(negative control)

Interpretation: see below
Irritation / corrosion parameter:
other: other: relative mean viability
Run / experiment:
15 min
Value:
99.2
Remarks on result:
no indication of irritation
Remarks:
Basis: mean. Time point: 15 min exposure + 42h expression. Reversibility: no data not applicable. Remarks: 100% = control. (migrated information)
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In vitro, the test item was non-irritant to the skin.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstituted human epidermis model.

The study design is based on international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46) and thestudy was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practice.

Preliminary tests were performed to detect the ability of the test item to directly reduce MMT as well as its coloring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in one main test. The test item, the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature during 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at 37°C, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissues viability which was set at 100% (reference viability).

In the preliminary test, the test item was presumed to have direct MTT reducing properties since the MTTsolution containing the test item changed color (brown color) when compared to the negative control. As a result, additional controls were performed on water-killed tissues in parallel to the main test.

The test item was found to have a coloring potential in the preliminary test since the water solution containing the test item changed color. As a result, additional controls were used in parallel to the main test for the evaluation of the non specific OD.

In the main test, following a 15-minute exposure and a 42-hour recovery period, the true relative mean viability of the tissues treated with the test item was 99.2%. This value being > 50%, the test item is therefore considered as non-irritant to skin.

 

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:
experimental study
Adequacy of study:
key study
Study period:
21 September 2011 - 13 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
the relative humidity recorded in the animal room was sometimes outside of the target ranges specified in the study plan.
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
idem above
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: breeder: Hypharm, La Corbière, Roussay, France
- Age at study initiation: at the beginning of the study, the animals were 2 to 4 months old
- Mean body weight at study initiation: a body weight of 3026 g and 3150 g, respectively
- Fasting period before study: no
- Housing: noryl cage
- Diet: free access to pelleted breeding diet "type 110C"
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: 26 October 2011 to 13 November 2011.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated right eye served as a control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: A dosage-volume of 0.1 g/animal was used.
Duration of treatment / exposure:
Not applicable: single application not followed by rinsing.
Observation period (in vivo):
1, 24, 48 and 72 h; if relevant, daily until reversibility of reactions
Number of animals or in vitro replicates:
2 males.
Details on study design:
REMOVAL OF TEST SUBSTANCE: No

SCORING SYSTEM: Draize scale.

- Conjunctival chemosis (lids and/or nictitating membranes):
0 no swelling
1 any swelling above normal (includes nictitating membranes)
2 obvious swelling with partial eversion of lids
3 swelling with lids about half-closed
4 swelling with lids more than half-closed

- Conjunctival redness (palpebral and bulbar conjunctivae, cornea and iris):
0 blood vessels normal
1 a number of blood vessels definitely hyperemic (injected)
2 diffuse, crimson colour, individual vessels not easily discernible
3 diffuse, beefy red

- Discharge:
0 absence of discharge
1 slight discharge (does not include small amounts normally found in inner canthus)
2 discharge with moistening of lids and hairs adjacent to lids
3 discharge with moistening of lids and hairs on wide area around the eye

- Iris lesions
0 normal
1 markedly deepened rugae, congestion, swelling, moderate circum-corneal hyperemia,or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 no reaction to light, haemorrhage, gross destruction (any or all of these)

- Cornea intensity of opacity (direct examination and, if necessary, with an UV lamp)
0 no ulceration or opacity
1 scattered or diffuse areas of opacity (other than slight dulling or normal lustre), details of iris clearly visible
2 easily discernible translucent area, details of iris slightly obscured
3 nacreous areas, no details of iris visible, size of pupil barely discernible
4 opaque cornea, iris not discernible through the opacity

- Cornea area of opacity (direct examination and, if necessary, with an UV lamp)
1 one quarter (or less) but not zero
2 greater than one quarter but less than a half
3 greater than one half but less than three quarters
4 greater than three quarters up to whole area

- Any other lesions observed were noted

TOOL USED TO ASSESS SCORE: UV lamp after instillation of 0.5% sodium fluorescein solution
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 24, 48 and 72 h (mean)
Score:
2.7
Max. score:
4
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
other: 24, 48 and 72 h (mean)
Score:
3
Max. score:
3
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 24, 48 and 72 h (mean)
Score:
1
Max. score:
1
Irritation parameter:
cornea opacity score
Remarks:
(intensity)
Basis:
animal #1
Time point:
other: 24, 48 and 72 h (mean)
Score:
2
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 24, 48 and 72 h (mean)
Score:
3
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
other: 24, 48 and 72 h (mean)
Score:
3
Max. score:
3
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: 24, 48 and 72 h (mean)
Score:
1
Max. score:
1
Irritation parameter:
cornea opacity score
Remarks:
(intensity)
Basis:
animal #2
Time point:
other: 24, 48 and 72 h (mean)
Score:
2.3
Max. score:
3
Interpretation of results:
Category 2A (irritating to eyes)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was irritant when administered by the ocular route to rabbits.
According to the criteria of CLP Regulation, the test item should be classified category 2 and assigned the signal word "warning" and the hazard statement "H319: causes serious eye irritation".
Executive summary:

The objective of this study was to evaluate the potential irritant properties of the test item for the eye following a single administration to rabbits.

This study was conducted in compliance with the principles of Good Laboratory Practice according to guideline OECD 404.

The test item was first administered to a single male New Zealand White rabbit. As ocular reactions were oserved, a second animal was tested. The test item was administered in the conjunctival sac of the left eye. The right eye remained untreated and served as control. A quantity of 0.1 g/animal was used. Just after the 1-hour scoring, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl). Each animal was observed once a day for mortality and clinical signs.Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions. The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal. Body weight was recorded on the day of treatment and the end of the observation period and sacrificed without macroscopic post-mortemexamination.

 

A chemosis (graded up to marked) was observed from day 1 to day 13 in the first animal and from day 1 to day 10 in the second animal.

A moderate to severe redness of the conjunctiva was observed in both animals, from day 1 to day 7 in the first animal and from day 1 to day 6 in the second animal. The redness was slight on day 8 in the first animal and on day7 in the second animal.

Iris lesions were noted from days 1 to 6 or or 5 in both animals. Corneal opacity was observed from days 2 to 5 in both animals. In addition, whitish purulent discharge was observed from days 2 to 6 in the first animal and from days 1 to 4 in the second animal.

Brownish area was observed on the conjunctiva from days 2 to 12 in the first animal and from days 6 to 11 in the second animal.

Mean scores calculated for each animal over 24, 48 and 72 hours were as follows:

.            chemosis: 2.7 and 3.0,

.            redness of the conjunctiva: 3.0 and 3.0,

.            iris lesions: 1.0 and 1.0,

.            corneal opacity: 2.0 and 2.3 for intensity; 1.7 and 1.7 for area

The test item was irritant when administered by the ocular route to rabbits.

According to the criteria of CLP Regulation,the test item should be classified category 2 and assigned the signal word "warning" and the hazard statement "H319: causes serious eye irritation".

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation / corrosion:

The potential of Dodecane-12-lactam, manufacturing of, by-products from, distillation residues to induce skin irritation was assessed using a sequential testing strategy. The purpose of this strategy was to avoid the unnecessary use of animals and to minimise any testing that is likely to produce severe responses in animals.

The objective of the first study performed in vitro was to determine whether or not the test item was corrosive.

I/The objective of this first GLP study (in vitro Episkin corrosion, CitoxLAB 2011) was to evaluate the corrosive potential of the test item, Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, using the EpiskinTMreconstituted skin model following OECD 431 guideline.

As the test item was found to have probable direct MTT reducing properties in the preliminary test, MTT reducing test item was applied to two water-killed tissues for each exposure time and two water-killed tissues remained untreated.

The non specific MTT reduction were 1%, 4% and 2% following the 3 minutes, 1 hour and 4 hours exposure durations, respectively. As a result, the corrected OD values of the test item treated water killed tissues were subtracted from the mean cOD of the test item treated living tissues to calculate the true amount of MTT reduction.

The relative mean viabilities of the test item treated tissues were : 3 minutes exposure: 117.3%,1 hour exposure: 113.4%, 4 hours exposure: 115.3%.

The blue discoloration of the test item treated tissues following the 3 minutes, 1 hour and 4 hours exposure periods was representative of viable tissue.

The test item,Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, tested in its original form, is considered to be non corrosive to the skin as the relative mean viabilities is >35%.

II/The objective of the second GLP study (in vitro Episkin irritation, CitoxLAB 2011) was to evaluate the skin irritation potential of the test item Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues, using the EpiskinTMreconstituted skin model following OECD 439 guideline.

In the main test, following a 15-minute exposure and a 42-hour recovery period, the true relative mean viability of the tissues treated with the test item was 99.2%. This value being > 50%, the test item is therefore considered as non-irritant to skin.

In vivo eye irritation:

The objective of this in vivo eye irritation study (Silvano, CitoxLAB, 2011) was to evaluate the potential irritant properties of the test item for the eye following a single administration to rabbits.This study was conducted in compliance with the principles of Good Laboratory Practice according to guideline OECD 404.

The test item was first administered to a single male New Zealand White rabbit. As ocular reactions were observed, a second animal was tested.The test item was administered in the conjunctival sac of the left eye. The right eye remained untreated and served as control. A quantity of 0.1 g/animal was used. Just after the 1-hour scoring, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl). Each animal was observed once a day for mortality and clinical signs. Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions. The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal.

 

A chemosis (graded up to marked) was observed from day 1 to day 13 in the first animal and from day 1 to day 10 in the second animal.

A moderate to severe redness of the conjunctiva was observed in both animals, from day 1 to day 7 in the first animal and from day 1 to day 6 in the second animal. The redness was slight on day 8 in the first animal and on day7 in the second animal.

Iris lesions were noted from days 1 to 6 or or 5 in both animals. Corneal opacity was observed from days 2 to 5 in both animals. In addition, whitish purulent discharge was observed from days 2 to 6 in the first animal and from days 1 to 4 in the second animal.

Brownish area was observed on the conjunctiva from days 2 to 12 in the first animal and from days 6 to 11 in the second animal.

Mean scores calculated for each animal over 24, 48 and 72 hours were as follows:

.            chemosis: 2.7 and 3.0,

.            redness of the conjunctiva: 3.0 and 3.0,

.            iris lesions: 1.0 and 1.0,

.            corneal opacity: 2.0 and 2.3 for intensity; 1.7 and 1.7 for area

The test item was irritant when administered by the ocular route to rabbits.


Effects on eye irritation: irritating

Justification for classification or non-classification

The relative mean viability of the tissues observed in the EpiskinTMreconstituted skin model being >50%, no classification is warranted for skin irritation under EU Dangerous Substances Directive 67/548/EEC or CLP EU Regulation 1272/2008.

On the basis of the results of the mean scores in the in vivo eye irritation study which were >2,3 and < 4 for erythema and according to the criteria of CLP Regulation,the test item should be classified category 2 and assigned the signal word "warning" and the hazard statement "H319: causes serious eye irritation".

Categories Display