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Description of key information

Two in vitro irritation tests are available (OECD 437 and OECD 439).

One in vivo eye irritation test was also performed (OECD 405)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 February 2013 - 25 February 2013
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)
Species:
other: reconstructed human epidermis
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
Episkin TM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C
Dates of experimental phase: from 12 February 2013 to 20 February 2013.
Type of coverage:
other: not applicable (in vitro)
Preparation of test site:
other: not applicable (in vitro)
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro negative and positive controls
Amount / concentration applied:
10 mg (± 2 mg) spread on tissues
Duration of treatment / exposure:
Exposure period of 15 minutes, followed by rinsing.
Observation period:
MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells.
Number of animals:
Not applicable
Triplicate for tested substance (test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Rinsing: At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. Excess D-PBS was removed by blotting the bottom of the tissue culture insert with absorbent paper.
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: Dulbecco’s Phosphate-Buffered Saline (D-PBS).

SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank

Interpretation: see below
Irritation parameter:
other: relative mean viability
Basis:
mean
Time point:
other: 15 min exposure + 42h expression
Reversibility:
no data
Remarks:
not applicable
Remarks on result:
other: 100% = control
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is considered to be non-irritant to skin.
According to the results of this study, the classification of the test item should be:
. not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008).
Executive summary:

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

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

 

Methods

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

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

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

 

Results

Preliminary tests

In the preliminary test, thetest item was found not to have direct MTT reducing properties.

 

The test item was found not to have a colouring potential in the preliminary test.

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

 

Following a 15 -minute exposure period and a 42-hour recovery period, the mean relative viability of the test item-treated tissues was 102% with a standard deviation of 9%.

 

Conclusion

The test item is considered to be non-irritant to skin.

According to the results of this study, the classification of the test item should be:

.  not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008). 

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:
26 February 2013 - 28 March 2013
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)
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
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: CEGAV, Argenvilliers, France
- Age at study initiation: 2 to 4 months old on the day of treatment
- Mean body weight at study initiation: 2833 g (range: 2715 g to 2990 g)
- Housing: the animals were individually housed in noryl cages
- Diet: 110 pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 6 days (first animal) or 4 days (the two other animals) 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.

IN-LIFE DATES: 18 March 2013 to 28 March 2013.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated right eye served as a control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL/animal.
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:
Three 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:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: day 3
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
1
Reversibility:
fully reversible within: day 4
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: day 4
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
cornea opacity score
Remarks:
(intensity)
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
cornea opacity score
Remarks:
(intensity)
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Irritation parameter:
cornea opacity score
Remarks:
(intensity)
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24, 28 and 72 h (mean)
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: CLP Regulation
Conclusions:
The test item was non irritant when administered by ocular route to rabbits.
Therefore, the test item should not be classified as irritating to the eyes according to the criteria of CLP Regulation.
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.

Methods

The test item was first administered to a single male New Zealand White rabbit.

 

As mean value from grading at 24, 48 and 72 hours after instillation was < 2 for conjunctival edema (chemosis) or for conjunctival redness and/or < 1 for iris lesion or for corneal opacity, the test item was administered in the left eye of two other animals.

 

The test item was administered inthe conjunctival sac of the left eye. The right eye remained untreated and served as control.

A dosage-volume of 0.1 mL/animal was used.

The eyes were not rinsedbefore 24-hour reading.

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 end of the observation period. 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 at the end of the evaluation of ocular reactions.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post‑mortem examination.

 

Results

No unscheduled deaths occurred during the study and no clinical signs were noted in any animals.

The body weight of the animals was unaffected by the test item treatment.

No ocular reactions were observed in the right untreated control eye.

In the left treated eye, slight chemosis of the conjunctiva was noted in all animals on day 1 and it persisted on day 2 in 1/3 males.

Slight or moderate redness of the conjunctiva was observed in all animals on days 1 and 2. Then, slight redness of the conjunctiva persisted on day 3 in 2/3 animals.

No iris and corneal lesions were noted in any animals.

 

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

.          chemosis : 0.0, 0.0 and 0.3 ; showing no significant eye irritation,

.          redness of the conjunctiva: 0.3, 0.7 and 1.0 ; showing no significant eye irritation,

.          iris lesions: 0.0, 0.0 and 0.0 ; showing no significant eye irritation,

.          corneal opacity: 0.0, 0.0 and 0.0 ; showing no significant eye irritation.

 

Conclusion

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

 

Therefore, the test item should not be classified as irritating to the eyes according to the criteria of CLP Regulation.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The registered substance is not irritating to the skin and eyes; not classified