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Diss Factsheets

Administrative data

Description of key information

 The substance was found to be neither corrosive nor irritant when tested in an in-vitro skin model.  Similarly,  the substance was considered to be non-corrosive to the eyes when tested on in-vitro rabbit eyes.
In-vivo, the substance was slightly irritant to skin and produced in eyes minimal transient conjunctival reactions within 1 hour after test treatment.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-11-02 to 2012-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: CEGAV, Argenvilliers, France
- Age/weight at study initiation: at the beginning of the study, the animals were 2 to 4 months old and had a mean body weight of 3850 g (range: 3695 g to 3975 g)
- Fasting period before study: no
- 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: 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.

IN-LIFE DATES: 27 November 2012 to 07 December 2012.
Type of coverage:
semiocclusive
Preparation of test site:
other: clipped
Vehicle:
unchanged (no vehicle)
Controls:
other: each animal served as its own control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 g/flank.
Duration of treatment / exposure:
3 min, 1 h, 4 h.
Observation period:
1, 24, 48 and 72 h after removal of the dressing and if relevant, daily until reversibility of reactions.
Number of animals:
3 males.
Details on study design:
TEST SITE
- Area of exposure: Right and left anterior and/or posterior flanks of the animals
- % coverage: 6 cm2
- Type of wrap if used: gauze pad held in place by a non-irritation semi-occlusive dressing and a restraining bandage.

REMOVAL OF TEST SUBSTANCE
- Washing:using a moistened cotton pad
- Time after start of exposure: at removal of each dressing (see Duration of exposure)

SCORING SYSTEM: according to OECD guideline 404

Irritation parameter:
erythema score
Remarks:
: 4-hour exposure
Basis:
animal #1
Time point:
other: 24, 48, 72 h (mean)
Score:
0.7
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
erythema score
Remarks:
: 4-hour exposure
Basis:
animal #2
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Remarks:
: 4-hour exposure
Basis:
animal #3
Time point:
other: 24, 48, 72 h (mean)
Score:
0
Max. score:
4
Irritation parameter:
edema score
Remarks:
: 4-hour exposure
Basis:
animal #1
Time point:
other: 24, 48, 72 h (mean)
Score:
0
Max. score:
4
Irritation parameter:
edema score
Remarks:
: 4-hour exposure
Basis:
animal #2
Time point:
other: 24, 48, 72 h (mean)
Score:
0
Max. score:
4
Irritation parameter:
edema score
Remarks:
: 4-hour exposure
Basis:
animal #3
Time point:
other: 24, 48, 72 h (mean)
Score:
0
Max. score:
4
Irritant / corrosive response data:
After a 3-minute and a 1-hour exposures, a very slight erythema (grade 1) was observed from day 1 to day 4 in the treated animal .
After a 4-hour exposure, a well defined erythema (grade 2) was noted in one animal and a slight erythema (grade 1) was noted in the two others one hour after patch removal. At the 24-hour reading, a slight erythema was still present in one animal but cleared at the 72-hour reading.

Table 7.3.1/1 Cutaneous reactions after a 4 -hour exposure

Rabbit number

Dermal irritation

SCORES

Mean irritation score (1)

1h

24h

48h

72h

D1

D2

D3

D4

A30281

Erythema

2

1

1

0

0.7

Edema

0

0

0

0

0.0

Other

*

*

*

*

 

A30282

Erythema

1

0

0

0

0.0

Edema

0

0

0

0

0.0

Other

*

*

*

*

 

A30283

Erythema

1

0

0

0

0.0

Edema

0

0

0

0

0.0

Other

*

*

*

*

 

(1)    mean of scores on days 2,3 and 4

h = hour

D= day

* = none

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance is not classified according to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.
Executive summary:

The potential of the test item to induce skin irritation was assessed in 3 rabbits according to OECD Guideline 404 in compliance with Good Laboratory Practices.

The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single New Zealand White rabbit. After the 4-hour application, since the mean value from grading at 24, 48 and 72 hours after patch removal was < 2.3 for erythema or for edema, the test item was applied on the skin of two other animals for 4 hours.

A quantity of 0.5 g/flank was applied to a skin area of approximately 6 cm2under a semi-occlusive dressing for 4 hours. Skin reactions were observed 1, 24, 48, 72 hours after removal of the dressing. The mean values of the scores for erythema and oedema were calculated for each animal. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded at the beginning and the end of the observation period. On completion of the observation period, the animals were used subsequently for the evaluation of the ocular irritation potential of the same test item.

No unscheduled deaths occurred during the study and no clinical signs indicative of systemic toxicity were noted in any animals.The body weight of the animals was unaffected by the test item treatment.

After a 4-hour exposure, a well defined erythema (grade 2) was noted in one animal and a slight erythema (grade 1) was noted in the two others one hour after patch removal. At the 24-hour reading, a slight erythema was still present in one animal but cleared at the 72-hour reading.

 The mean scores calculated for each animal over 24, 48 and 72 hours were 0.7, 0.0, 0.0 for erythema and 0.0, 0.0; 0.0 for oedema.

 It was concluded that the test item was slightly irritant when applied topically to rabbits.

 

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:
from 2012-11-02 to 2012-12-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: 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:
TEST ANIMALS
- Source: breeder: CEGAV, Argenvilliers, France
- Age/weight at study initiation: the animals were between 3 and 5 months old (i.e. between 17 and 18 weeks old) at the beginning of the study and their body weight exceeded 3.5 kg for all animals (range : 3.795 kg to 4.115 kg).
- Fasting period before study: no
- 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: 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.

IN-LIFE DATES: 12 December 2012 to 24 December 2012.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated right eye served as a control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: a quantity 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; since all animals showed no evidence of irritation after 72 hours, the evaluation of ocular reactions was stopped.
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, if applicable.

TOOL USED TO ASSESS SCORE: UV lamp after instillation of 0.5% sodium fluorescein solution
Irritation parameter:
overall irritation score
Basis:
animal #1
Time point:
other: 24,48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
overall irritation score
Basis:
animal #2
Time point:
other: 24,48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
overall irritation score
Basis:
animal #3
Time point:
other: 24,48 and 72 hours
Score:
0
Max. score:
4
Irritant / corrosive response data:
No ocular reactions were observed in the right untreated control eye.
In the left treated eye, slight redness of the conjunctiva (grade 1) was observed on day 1 in all animals within 1 hour after test treatment.
Then, no other ocular reactions were observed in any animals during the study period.
Other effects:
None
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance is not classified according to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.
Executive summary:

The potential of the test item to induce eye irritation was assessed in rabbits according to OECD Guideline 405 in compliance with Good Laboratory Practice.

A volume of 0.1 mL of the test substance was placed into the conjunctival sac of the left eye of a single New Zealand White rabbits. The right eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made 1, 24, 48 and 72 hours following treatment according to the Draize scoring method. As mean value from grading at 24, 48 and 72 hours after instillation was < 2 for conjunctival edema (chemosis) and for conjunctival redness, < 1 for iris lesion and for corneal opacity, the test item was administered in the left eye of two other animals.

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 sacrifice of animals. 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.

 

No unscheduled deaths occurred during the study and no clinical signs indicative of systemic toxicity were noted in any animals.The body weight of the animals was unaffected by the test item treatment.

Slight redness of the conjunctiva was observed in all animals on day 1 within 1 hour after test treatment. Then, no other ocular reactions were observed in any animals during the study period.

It was concluded that the test item required no classification according to regulation (EC) n°1272/2008.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion:

The potential of the substance 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.

This key study was conducted in compliance with OECD Guideline No. 431 and the principles of Good Laboratory Practices. The Episkin reconstituted human epidermis was the in vitro model selected. The test item, and both negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 4 hours; test item for 3 minutes, 60 minutes and 4 hours. At the end of the designated incubation periods, the cell viability was assessed by means of the colourimetric MTT reduction assay. The relative mean viabilities of the test item-treated tissues were 91% , 92% and 104% of the negative control for 3, 60 and 240 minutes exposure periods respectively. As the mean viability was above the cut-off value of 35% whatever the exposure duration, the test item was considered to be non corrosive to the skin (Valin, 2012).

In the second in vitro study, the potential of the test item to induce irritation was assessed.

This key study was conducted in compliance with OECD Guideline No. 439 and the principles of Good Laboratory Practices. The Episkin reconstituted human epidermis was used as skin model. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, tissues were rinsed and incubated for 42 hours at 37°C. The cell viability was then assessed by means of the colourimetric MTT reduction assay. The relative mean viability of the tissues treated with the test item was 108% with a standard deviation of 2%. As the mean relative viability was above 50%, the concentration of the inflammatory mediator IL-1α in the culture medium was determined by ELISA

The mean concentration of IL-1α was 24.6 pg/mL . Due to this value being below the cut-off value of 60 pg/mL, the test item was predicted to be non-irritant to skin (Gerbeix, 2012a).

The result obtained in vitro was then confirmed in an in vivo test using only one animal at the beginning. This key study was conducted in compliance with OECD Guideline 404 and the principles of Good Laboratory Practices. The test item was applied for 3 minutes on the skin of a single animal. As no full thickness destruction of skin tissue was observed in the first hour after removal of the pad, the test item was applied on another treatment site for 1 hour. As no full thickness destruction of skin tissue was observed in the first hour after removal of the pad, the test item was applied on another treatment site for 4 hours.  After the 4-hour application on the animal, as mean value from grading at 24, 48 and 72 hours after patch removal was below 2.3 for erythema or for oedema, the test item was applied to the skin of two other animals for 4 hours. No unscheduled deaths occurred during the study and no clinical signs indicative of systemic toxicity were noted in any animals.The body weight of the animals was unaffected by the test item treatment. After a 4-hour exposure, a well defined erythema (grade 2) was noted in one animal and a slight erythema (grade 1) was noted in the two others one hour after patch removal. At the 24-hour reading, a slight erythema was still present in one animal but cleared at the 72-hour reading. The mean scores calculated for each animal over 24, 48 and 72 hours were 0.7, 0.0, 0.0 for erythema and 0.0, 0.0; 0.0 for oedema.  It was concluded that the test item was slightly irritant when applied topically to rabbits (Papineau, 2013a).

Eye irritation / corrosion:

The potential of the substance to induce eye 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 this first study performed in vitro was to determine whether or not the test item was corrosive.

This key study was conducted in compliance with OECD Guideline No. 437 and the principles of Good Laboratory Practices. Corneas obtained from freshly slaughtered calves were used as in vitro model. The test item, and both negative and positive controls were applied in triplicate on corneas and incubated for 4 hours. Before the treatment, a first opacity measurement was performed using an opacitometer.

At the completion of the treatment period, the test item was removed and a second opacity measurement was then performed. After this measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. After incubation, the optical density of the solution from the posterior chamber was measured in order to determine the permeability of the cornea.

The In Vitro Irritancy Score (IVIS) of the test item was 2.2. As the test item induced an IVIS below 55.1, it was considered to be non corrosive to the eye but according to the OECD Guideline 437, further testing should be conducted for classification and labeling purposes (Gerbeix, 2012b).

A follow-up in-vivo study was conducted to evaluate the acute eye irritation potential of the test substance in New Zealand White rabbits. This key study was conducted according to OECD Guideline 405 in compliance with the principles of Good Laboratory Practices.

A volume of 0.1 mL of the test substance was placed into the conjunctival sac of the left eye of a single New Zealand White rabbits. The right eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made 1, 24, 48 and 72 hours following treatment according to the Draize scoring method.As mean value from grading at 24, 48 and 72 hours after instillation was < 2 for conjunctival edema (chemosis) and for conjunctival redness, < 1 for iris lesion and for corneal opacity, the test item was administered in the left eye of two other animals.

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 sacrifice of animals. 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.

 No unscheduled deaths occurred during the study and no clinical signs indicative of systemic toxicity were noted in any animals. The body weight of the animals was unaffected by the test item treatment. Slight redness of the conjunctiva was observed in all animals on day 1 within 1 hour after test treatment.Then, no other ocular reactions were observed in any animals during the study period.

It was concluded that the test item required no classification according to regulation (EC) n°1272/2008 (Papineau, 2013b).


Justification for selection of skin irritation / corrosion endpoint:
Key study

Justification for selection of eye irritation endpoint:
Key study

Justification for classification or non-classification

Based on the results from in vitro and in vivo studies, the substance is not classified as a skin or eye irritant according to regulation EC No. 1272/2008

and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.