Registration Dossier

Administrative data

Description of key information

Skin:

The substance was not significantly irritating for the skin in a Episkin irritation assay in vitro. An older in vivo assay evidenced that it did not warrant classification for skin irritation. According to ECVAM (2009) consensus, the results of the Episkin assay carried out in 2010 are, alone, sufficient to exclude skin irritation without need for in vivo data.

 Eye:

The substance was slightly irritating for the eye in vitro, which is the minimal possible grade in this test (there is no grade “non irritant”).Two older in vivo assays evidenced that it did not warrant classification for eye irritation.


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:
From 16 June 2010 to 10 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: pending to search
Deviations:
not applicable
Principles of method if other than guideline:
The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 Hhour post exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end point will be used to either confirm a non-irritant result or will be used to override the non irritant result.
GLP compliance:
yes (incl. certificate)
Species:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 10 µl of the test material was applied to the epidermis surface (to ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre)).
After 7 minutes contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period.
Duration of treatment / exposure:
15 minutes
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.
Irritation / corrosion parameter:
other:
Value:
97.8
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Reversibility: other: not applicable. (migrated information)
Other effects / acceptance of results:
The relative mean viability of the test material treated tissues was 97.8% after a 15 minute exposure.
The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material:

Material

OD540 of tissues

Mean OD540 of triplicate tissues

± SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Material

0.687

0.687

0.007

100.0

100*

1.0

0.681

99.1

0.694

101.0

Positive Control Material

0.105

0.067

0.035

15.3

9.7

5.1

0.060

8.7

0.035

5.1

Test Material

0.654

0.672

0.030

95.2

97.9

4.4

0.656

95.5

0.707

102.9

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Following the 15-Minute exposure period the test material treated tissues appeared blue which was considered indicative of viable tissue.

Due to the absence of cytotoxicity in the MTT assay it was necessary to determine the concentration ofinflammatory mediator IL-1α in the retained culture medium for confirmatory purposes.

The concentration ofinflammatory mediator IL-1α in the culture medium retained from the test material treated tissues was 27.273 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤ 18%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥ 0.6 and the SD value of the percentage viability was ≤ 18%. The negative control acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: other: see table above (any other information on materials and methods)
Conclusions:
The test material was considered to be Non-Irritant. This result was confirmed by assessment of the inflammatory mediator IL-1α.
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hhour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 97.8% after a 15-Minute exposure period.

Due to the absence of cytotoxicity in the MTT assay it was necessary to determine the concentration of inflammatory mediator IL-1α in the retained culture medium for confirmatory purposes.

The concentration of inflammatory mediator IL-1α in the culture medium retainedfrom the test material treated tissues was 27.273 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: 

The test material was considered to be Non-Irritant. This result was confirmed by assessment of the inflammatory mediator IL-1α.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 May 2010 to 16 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The method used is adapted from that described by Gautheron P. & al. (1992) Fundam. Appl. Toxicol. 18 442-449.
The principle of this evaluation is based on the measurement of two factors: the opacity and the permeability of the treated corneas. The changes in these parameters correspond to the damages induced to the tissues.
GLP compliance:
yes (incl. certificate)
Species:
other: isolated cornea
Strain:
other: bovine calf
Details on test animals or tissues and environmental conditions:
TEST CORNEAS
- Source: calf eyes were obtained from freshly slaughtered calves at the abattoir SOCAVIA, Cany Barville, France.

- Reason for choice: calf corneas are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

- Transport from supplier to CIT: the eyes were transported to CIT at ambient temperature, immerged in buffered Hanks medium containing antibiotics (Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin).

- Preparation of the corneas: the corneas were prepared as quickly as possible after receipt. Each step was carried out avoiding to touch the corneas in order to not injure them.

- Selection: upon arrival at CIT, all eyes were carefully examined macroscopically for defects (opacity, scratches, pigmentation, etc) and those exhibiting any defect were discarded. The too large eyes were also discarded in order to avoid the formation of folds at the assembly of corneas in the holder. The examination was performed under a lamp and using HBSS in order to maintain the corneas moistened and shiny. Each cornea was observed with attention, while making swivel the eye in order to see any less refringent areas under the light or any scratches.

- Preparation of the selected corneas: the tissue surrounding the eyeball was carefully pulled away and the cornea was dissected such that approximately 2 to 3 mm of sclera was present around the cornea. The isolated corneas were stored in HBSS until all corneas were dissected.
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a volume of 750 µL ± 8 µL was gently applied to the cornea, as uniformly as possible
Duration of treatment / exposure:
30 minutes and 4 hours
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable (three corneas were used for each treated series (test item, positive control and negative control)).
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): As the dosage form was liquid, the anterior compartment of the holder was emptied using a metal gavage tube attached to a vacuum pump, then the compartment was filled with heated cMEM (32°C). The dosage form having adhered to the walls of the compartment was eliminated using a cotton bud. Then the compartment was filled with heated cMEM (32°C). The rinsing was repeated 10 and 4 times in the 30-minute and 4-hour treatment, respectively (i.e. until the dosage form is completely removed from the compartment).


TOOL USED TO ASSESS SCORE: opacitometer, spectrophotometer, fluorescein
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value
Value:
0.7
Other effects / acceptance of results:
Following the 4-hour treatment, the mean in vitro score was -0.9.
No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4-hour treatment.
No notable opaque spots or irregularities were observed on test item-treated corneas, either following the 30-minute treatment or following the 4 hour treatment.

For each experiment, the acceptancecriteria were fulfilled:

.  the individual corneal opacity values of negative controls were < 10 in both experiments,

. the individual OD490 nm values of negative control corneas were < 0.100 in both experiments,

. the solution of fluoresceine (at 5 mg/mL in DPBS) diluted 1:1000 in cMEM had OD490 nm values between 0.850 and 0.940 in both experiments,

. following the 30-minute treatment, the positive control mean in vitro score was 90.3, thus demonstrating the sensitivity of the test system under the experimental conditions of this study.

Interpretation of results:
slightly irritating
Conclusions:
Under the experimental conditions of this study, according to both mean in vitro scores of the 30 minute and 4-hour treatments, the test item BAB tested in its original form is classified as slightly irritant for the isolated calf cornea.
Executive summary:

Method

The corneas were obtained from the eyes of freshly slaughtered calves at the abattoir. They were mounted in the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. Both compartments of the corneal holder were filled in excesswith Minimal Essential Medium Eagle completed with 1% fetal calf serum plus penicillin/streptomycin (cMEM), then the holders were preincubated for 1 hour at 32°C.

 

Three corneas were used for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed using an opacitometer (determining the light transmission through the center of each mounted cornea).

 

For the treatment, the test item was used in its original form.

 

The test item was tested sequentially in two consecutive experiments.

As the mean in vitro score at the 30-minute treatment was ≤ 10, the second experiment was undertaken using a 4-hour treatment.

 

At the completion of the treatment period, the test item was removed from the front opening of the anterior part of the holder and the epithelium was washed.

 

Following the 30-minute treatment, the corneas were incubated for 2 hours at 32°C. At the completion of the 2-hour incubation period, the second opacity measurement was performed.

Following the 4-hour treatment, the second opacity measurement was performed immediately without any further incubation after the rinsing of the dosage form.

 

After the second opacity measurement, the medium was removed from both compartments of each holder. The posterior compartment was refilled with cMEM at, while the anterior compartment received 1 mL of a 5 mg/mL fluoresceine solution in Dulbecco's Phosphate-Buffered Saline (DPBS). Then, the holders were incubated vertically for 90 minutes at 32°C.

 

At the end of the 90-minute incubation, the optical density of the solution from the posterior compartment of the holder was measured at 490 nm in order to determine the permeability of the cornea. Then the cornea was removed from the holder and observed for opaque spots and other irregularities.

Results

For each experiment, the acceptance criteria were fulfilled and the study was therefore considered to be valid.

 

No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4-hour treatment.

No notable opaque spots or irregularities were observed on test item-treated corneas, either following the 30-minute treatment or following the 4-hour treatment.

Following the 30-minute treatment, the mean in vitro score was 0.7. Then following the 4-hour treatment, the mean in vitro score was -0.9.

 

Conclusion

Under the experimental conditions of this study, according to both mean in vitro scores of the 30-minute and 4-hour treatments, the test item BAB tested in its original form is classified as slightly irritant for the isolated calf cornea.

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

Based on the results of the available data and studies on skin irritation, the substance has not to be classified for this hazard class following the classification criteria of the EU Regulation 12/2008 (CLP Regulation).

Based on the results of the available data and studies on eye irritation, the substance has not to be classified for this hazard class following the classification criteria of the EU Regulation 12/2008 (CLP Regulation).

Regarding respiratory irritation, there are no data available. Based on the non-irritant behaviour of the substance, both in skin and eyes, it is not expected that the substance could be a respiratory irritant.