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Endpoint summary

Description of key information

To date, two different studies on skin irritation/corrosion of FAT40851/A are available:
Mallaun (2010): In an in vivo skin irritation study in rabbits according to OECD Guideline 404 and EU Method B.4, FAT40851/A TE is not irritating to skin.
Heppenheimer (2010): In an in vitro skin corrosion study using a human skin model test according to OECD Guideline 431 and EU Method B.40, FAT40851/A was found to be not corrosive to skin.
In an in vitro BCOP test according to OECD Guideline 437, FAT40851/A was found to be not corrosive to the eyes. Applying the criteria of INVITTOX protocol no. 98, FAT40851/A can be judged to be a mild eye irritant. No study on respiratory irritation is available to date.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 12 November 2009; Experiment completion date - 13 November 2009; Study completion date - 01 December 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 431 (In vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9% all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Remarks:

Deionised water

Details on test system:

EST-1000 kits were purchased from CellSystems® Biotechnologievertrieb GmbH (53562 St. Katharinen; Germany). The EST-1000 tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EST-1000 tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅).
EST-1000 kits were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (November 10, 2009) EST-1000 tissues were kept in the refrigerator at 2 - 8 °C until November 12, 2009 prior to use. At least one hour before starting the assay, tissues were transferred to 6-well plates with assay medium, which is immediately replaced before the test is started.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test material was not crushed or ground in a mortar and a pestle since this did not improve the consistency. About 25 mg of the test material were wetted with 50 μL deionised water. The test item was spread to cover the surface of the tissue. For the positive and negative controls, a volume of 50 μL was dosed per tissue.

Duration of treatment / exposure:

3 minutes and 1 hour.

Number of replicates:

Two

Species:

human

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper.
- Time after start of exposure: after 3 min and 1h exposure

SCORING SYSTEM: MTT assay
After the exposure procedure, the cell culture inserts were incubated for 3 hours with MTT solution. Subsequently, the MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. The inserts were transferred into new 24-well plates and extracted with isopropanol for 17 hours 35 minutes.
Aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate and optical density (OD) was read at 570 nm (OD570).
The mean values were calculated for each set of 3 wells per tissue insert.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean Experiment I & II - 3 min

Value:

93.9

Negative controls validity:

valid

Remarks:

100% tissue viability

Positive controls validity:

valid

Remarks:

5.7% tissue viability

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean Experiment I & II - 1 hour

Value:

88.8

Negative controls validity:

valid

Remarks:

100% tissue viability

Positive controls validity:

valid

Remarks:

4.6% tissue viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The optical evaluation of the MTT-reducing capacity of the test item after a 1 hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

After exposure to the test item test substance the relative absorbance values were irrelevantly decreased to 93.9% after 3 minutes. After the 1 hour exposure relative absorbance values were reduced to 88.8%. Both values did not exceed the threshold for corrosivity of 50% for the 3 minutes exposure and 15% for the 1 hour exposure.

Therefore, the test item was not considered to be corrosive.

The optical evaluation of the MTT-reducing capacity of the test item after a 1 hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

Table 1: Results after treatment with test substance

Dose group	Exposure time	OD570		Mean absorbance	Rel. absorbence [% of negative control]
		Tissue 1 *	Tissue 2 *
Negative control	3 min	1.869	1.484	1.676	100.0
Positive control	3 min	0.089	0.100	0.095	5.7
test substance	3 min	1.635	1.514	1.574	93.9
Negative control	1 h	1.696	1.939	1.817	100.0
Positive control	1 h	0.083	0.085	0.084	4.6
test substance	1 h	1.451	1.778	1.614	88.8

* Mean of three replicate wells after blank correction

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was non corrosive to skin.

Executive summary:

This in vitro study was performed according to OECD Guideline 431 and to EU Method B.40 under GLP to assess the corrosive potential of test substance by means of the Human Skin Model Test.

 

Independent duplicate tissues of the human skin model EST-1000 were exposed to either the test item, the negative control or the positive control for 3 minutes and 1 hour, respectively. About 25 mg of the test item were applied to each tissue, wetted with 50 μL deionised water, and spread evenly over the surface of the tissue. A volume of 50 μL of either the negative control (deionised water) or the positive control (8.0 N KOH) was applied to each tissue. Viability of the cells were determined by MTT assay by measuring the absorbance at 570 nm (OD₅₇₀) and expressed as relative absorbance in % of negative control.

 

After exposure to the negative control the absorbance values exceeded the required acceptability criterion of mean OD₅₇₀ ≥0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period thus confirming the validity of the test system.

After exposure to the test item, the relative absorbance values were decreased to 93.9% after 3 minutes. After the 1 hour exposure relative absorbance values were reduced to 88.8%. Both values did not exceed the threshold for corrosivity of 50% for the 3 minutes exposure and 15% for the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

 

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item test substance was non corrosive to skin.

According to the referred classification (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), test substance does not have to be classified with respect to skin corrosion.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 03 February 2010; Experiment completion date - 23 February 2010; Study completion date - 16 March 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS Young Adult New Zealand White Rabbit, SPF
- Source: Charles River Deutschland GmbH Stolzenseeweg 32-36 88353 Kisslegg / Germany
- Age at study initiation: 15 weeks (male), 14 weeks (females)
- Weight at study initiation: 3026 g (male), 2852 - 3263 g (females)
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Haysticks and wood blocks were provided for gnawing.
- Diet: Pelleted standard Kliba Nafag 3418 rodent maintenance diet available ad libitum.
- Water: Community tap water from Füllinsdorf ad libitum.
- Acclimation period: 03-Feb-2010 to 07-Feb-2010 (one female), 03-Feb-2010 to 08-Feb-2010 (one male and one female)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): relative humidity between 30-70 % (values above 70 % during cleaning process possible),
- Air changes (per hr): Air-conditioned with 10-15 air changes per hour
- Photoperiod: automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied : 0.5 g/animal

VEHICLE
- Amount(s) applied (volume or weight with unit): The test item was moistened with approximately 0.5 mL of purified water before application

Duration of treatment / exposure:

4h

Observation period:

14d

Number of animals:

1 male and 2 females

Details on study design:

TEST SITE
- Area of exposure: approximately 100 cm² (10 cm x 10 cm). Four days before treatment, the left flank was clipped with an electric clipper The skin of the animals was examined one day before treatment, and regrown fur of all animals was clipped again.
- % coverage: A surgical gauze patch (ca. 2.5 cm x 2.5 cm) held in contact with the skin by means of an adhesive hypoallergenic aerated semiocclusive dressing and a restrainer bandage wrapped around the abdomen.

REMOVAL OF TEST SUBSTANCE
After 4 hours exposure time, the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site.

SCORING SYSTEM:
The skin reaction was assessed according to the numerical scoring system listed in the Commission Regulation (EC) No. 440/2008, B.4, at approximately 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after exposure (removal of the dressing, gauze patch and test item). The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. An animal is positive when the mean score is 2.3 or greater. The test is positive for irritation when at least 2 animals are positive for the same endpoint (erythema/eschar or edema).

GRADING OF SKIN REACTIONS
Erythema and Eschar Formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) or eschar formation (injuries in depth preventing erythema reading) 4

Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (edges raised approximately 1 mm) 3
Severe edema (raised more than 1 mm and extending beyond the area of exposure) 4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

IRRITATION
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for edema grades, separately. The individual mean score for erythema/eschar and edema for each of the three animals was 0.

CORROSION
Neither alterations nor corrosive effects were observed on the treated skin.

Other effects:

VIABILITY / MORTALITY
No intercurrent deaths occurred during the course of the study.

CLINICAL SIGNS
No clinical signs were observed throughout the entire observation period.

BODY WEIGHTS
The body weight of the animals was within the range commonly recorded for this strain and age.

SKIN REACTIONS
Slight orange staining of the treated skin produced by the test item was noted in all three animals from the 1-hour observation after removal of the dressing and persisted up to 10 days in the male and up to 14 days (end of the observation period) in both females.

Interpretation of results:

GHS criteria not met

Conclusions:

The individual mean score for erythema/eschar and oedema for each of the three animals was 0.
According to the referred classification (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), test substance does not have to be classified in respect to the skin irritation study in rabbits.

Executive summary:

The primary skin irritation potential of test substance was investigated according to OECD test guideline No. 404 and Commission Regulation (EC) No. 440/2008, B.4 under GLP. The test item was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after removal of the dressing. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The 24/48/72h mean score for erythema/eschar and oedema was found to be 0. The application of test substance to the skin resulted in no signs of irritation. Slight orange staining of the treated skin caused by the test item was observed in all animals. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed. According to the referred classification (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), test substance does not have to be classified with respect to skin irritation in rabbits.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start and completion date - 17 November 2009; Study completion date - 22 January 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay"

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Species:

cattle

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes were collected from the abattoir and transported in Hank’s BSS supplemented with streptomycin / penicillin at room temperature. The corneae were isolated immediately after delivery of the eyes to the laboratory.

PREPARATION OF CORNEA
All eyes were carefully examined macroscopically for defects and discarded when indicated. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects. Each cornea was mounted in a cornea holder; subsequently, both compartments of the holder were filled with complete medium and allowed to equilibrate for about 1 hour at 32 ± 2 °C in a water-bath.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

ca 100 mg test item

Duration of treatment / exposure:

240 min (± 5 min)

Number of animals or in vitro replicates:

Number of Corneae per Group: 3
Number of Test Item Group: 1
Number of Negative Control Group: 1
Number of Positive Control Group: 1
Total Number of Corneae: 9

Details on study design:

Study design
The corneae were distributed as follows:
Negative Control: 3
Positive Control: 3
Test Item: 3
Fresh MEM was placed in the posterior compartment, while the anterior compartment received the test item (approx. 100 mg) or negative or positive control at a volume of 1.0 mL each on the surface of the corneae and was incubated at 32 ± 2 °C in the water-bath in a vertical position.
The positive control was 10 % (w/v) Benzalconium chloride. 0.9 % (w/v) saline was used as negative control item. The incubation time lasted 240 minutes (± 5 minutes). After the test item or control items, respectively, were rinsed off from the application side by changing cMEM several times, fresh cMEM was replaced in both compartments and opacity was measured (t 240). In the second step of the assay, permeability of the cornea possibly caused by the test item, was determined. Fresh complete medium was added to the posterior compartment and 1 mL of a Na-fluorescein solution, 0.5 % dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for further 90 minutes at 32 ± 2 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. The opacitometer was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. After recording the basal opacity of all corneae, the values of all corneae were noted. Sets of three corneae were used for treatment with the test item and the negative and positive controls. Complete medium was completely removed from the anterior compartment and replaced by the test item, positive or negative control. The anterior compartment was plugged. The cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a water-bath at 32 ± 2 °C for 240 minutes. Afterwards, the opacity was measured again (t 240).

Permeability Determination
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5 % (w/v) fluorescein solution. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 2 °C. Complete medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 experiments

Value:

19.34

Negative controls validity:

valid

Remarks:

Mean invitro irritation score 2.59

Positive controls validity:

valid

Remarks:

Mean invitro irritation score 221.84

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item FAT 40851/A did not cause any permeability of the corneae compared with the results of the negative control. Due to the dying property of the test item the corneae were coloured but still translucent after the treatment period, thus the increase of the opacity value compared with the results of the negative control is most probably caused by the dying effect. The calculated mean in vitro score was 19.34 and therefore, the test item has to be classified as mild eye irritant. But the actual effect of the present test might be weaker than the irritation score indicates.

With the negative control (0.9 % NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro score was calculated as 2.59. The positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The mean in vitro score was calculated as 221.84. The test item did not cause any permeability of the corneae compared with the results of the negative control. Due to the dying property of the test item the corneae were coloured but still translucent after the treatment period, thus the increase of the opacity value compared with the results of the negative control is most probably caused by the dying effect. The calculated mean in vitro score was 48.90 and therefore, the test item has to be classified as moderate eye irritant according to INVITTOX protocol no. 98, but the actual effect of the present test might be weaker than the irritation score indicates. 

Results after 240 Minutes Incubation Time

Test group	Opacity value		Permeability		In vitro score	Mean in vitro score	Proposed in vitro irritation scale
		Mean		Mean
Negative control	0	0.33	0.116	0.150	1.74	2.59	Non eye irritant
	1		0.147		3.20
	0		0.188		2.82
Positive control	244.7 *	213.7	0.546 *	0.545	252.86	221.84	Very severe eye irritant
	191.7 *		0.603 *		200.72
	204.7 *		0.486 *		211.95
test substance	24.7 *	19.0	0.031 *	0.022	25.13	19.34	Mild eye irritant
	25.7 *		0.025 *		26.04
	6.67 *		0.012 *		6.84

* corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

In this study and under the experimental conditions reported, the test item is considered to be a mild eye irritant according to the criteria proposed in the INVITTOX protocol no. 98 and causes no irreversible effects to the eyes.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of test substance by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437 and INVITTOX protocol no. 98 under GLP. After a first opacity measurement of the fresh bovine corneae (t0), the test item test substance, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 2 °C in cMEM medium supplemented with sodium bicarbonate and L-glutamine and 1 % fetal calf serum (FCS) (complete medium). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240). After the opacity measurements permeability of the corneae was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °C in a horizontal position. The coming out liquid was measured spectrophotometrically. With the negative control (0.9 % NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (10 % (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The test item test substance did not cause any permeability of the corneae compared with the results of the negative control. Due to the dying property of the test item the corneae were coloured but still translucent after the treatment period, thus the increase of the opacity value compared with the results of the negative control is most probably caused by the dying effect. The calculated mean in vitro score was 19.34 and therefore, the test item has to be classified as mild eye irritant. But the actual effect of the present test might be weaker than the irritation score indicates. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item test substance is considered to be a mild eye irritant according to the criteria proposed in the INVITTOX protocol no. 98. Based upon the classification criteria according to „Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008“ the test item is not classified as Eye Effects Category 1 (irreversible effects, eye corrosion).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 08 June 2011; Experiment start date - 09 June 2011; Experiment completion date - 20 June 2011; Study completion date - 19 September 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: NIER notification No. 2010-29, Korea

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: SAMTAKO BIOKOREA CO., LTD.
- Age at study initiation: Approx. 3 months
- Weight at study initiation: 1945.8 to 2088.0 g
- Housing: In stainless steel cages [380(W) x 480(D) x 300(H) mm, Jeongdo Ltd., Korea]
- Diet: Pellet diet for rabbit ad libitum
- Water: Filtered and UV irradiated water ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±3
- Humidity (%): 50 ±20
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): approx. 12/12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g

Duration of treatment / exposure:

Once the test item was instilled into the conjunctivae sac of the eye, the lids were gently held together for approx. 1 second not to leak the test item.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:

Cornea
Opacity: degree of density (readings should be taken from most dense area)*
- No ulceration or opacity ............................................................................................................................ 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible....... 1
- Easily discernible translucent areas. Details of iris slightly obscured.............................................................. 2
- Nacrous area; no details of iris visible; size of pupil barely discernible............................................................ 3
- Opaque cornea; iris not discernible through the opacity................................................................................. 4

Iris
- Normal...................................................................................................................................................... 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish
reaction is considered to be an effect)............................................................................................................ 1
- Hemorrhage, gross destruction, or no reaction to light.................................................................................... 2

Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)
- Normal ······························································································································ 0
- Some blood vessels hyperaemic (injected) ······················································· 1
- Diffuse, crimson colour; individual vessels not easily discernible ············· 2
- Diffuse beefy red ·········································································································· 3

Chemosis
Swelling (refers to lids and/or nictating membranes)
- Normal ······························································································································ 0
- Some swelling above normal ·················································································· 1
- Obvious swelling, with partial eversion of lids ················································· 2
- Swelling, with lids about half closed ···································································· 3
- Swelling, with lids more than half closed ·························································· 4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

mean 4 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

4 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

mean of 4 animals

Time point:

24/48/72 h

Score:

0.58

Max. score:

3

Reversibility:

fully reversible within: 72h

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

mean of 4 animals

Time point:

24/48/72 h

Score:

0.08

Max. score:

4

Reversibility:

fully reversible within: 48h

Remarks on result:

no indication of irritation

Other effects:

No mortality was noted during the course of the study.
No treatment-related clinical signs were observed in any treated animals.
All tested animals showed normal gains in body weights.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results and according to the Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, FAT 40851/B does not have to be classified as eye irritant.

Executive summary:

The primary eye irritation potential of FAT 40851/B was investigated according to OECD 405. The test item was applied by instillation of 0.1 g into the right eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed after 1, 24, 48 and 72 hours after test item instillation. The instillation of FAT 40851/B into the eye resulted in slight early-onset and transient occular changes, such conjunctival reddening, chemosis and discharge. The mean 24/48/72 h corneal opacity, iris, conjunctivae and chemosis scores were 0, 0, 0.58 and 0.08 respectively. The conjunctivae and chemosis was reversible withing 72 h. No abnormal findings were observed in the cornea or iris of any animal at any of the examination. Based on the results and according to the Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, FAT 40851/B does not have to be classified as eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosion (OECD 431)

This in vitro study was performed according to OECD Guideline 431 and to EU Method B.40 under GLP to assess the corrosive potential of test substance by means of the Human Skin Model Test.

Independent duplicate tissues of the human skin model EST-1000 were exposed to either the test item, the negative control or the positive control for 3 minutes and 1 hour, respectively. About 25 mg of the test item were applied to each tissue, wetted with 50 μL deionised water, and spread evenly over the surface of the tissue. A volume of 50 μL of either the negative control (deionised water) or the positive control (8.0 N KOH) was applied to each tissue. Viability of the cells were determined by MTT assay by measuring the absorbance at 570 nm (OD570) and expressed as relative absorbance in % of negative control. After exposure to the negative control the absorbance values exceeded the required acceptability criterion of mean OD570≥ 0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1-hour exposure period thus confirming the validity of the test system. After exposure to the test item test substance the relative absorbance values were decreased to 93.9 % after 3 minutes. After the 1-hour exposure relative absorbance values were reduced to 88.8 %. Both values did not exceed the threshold for corrosivity of 50% for the 3 minutes exposure and 15% for the 1-hour exposure. Therefore, the test item was not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item test substance was non-corrosive to skin. According to the referred classification (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), test substance does not have to be classified with respect to skin corrosion.

 

In vivo skin irritation (OECD 404)

The primary skin irritation potential of test substance was investigated according to OECD test guideline No. 404 and Commission Regulation (EC) No. 440/2008, B.4 under GLP. The test item was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after removal of the dressing. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The 24/48/72h mean score for erythema/eschar and oedema was found to be 0. The application of test substance to the skin resulted in no signs of irritation. Slight orange staining of the treated skin caused by the test item was observed in all animals. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed. According to the referred classification (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), test substance does not have to be classified with respect to skin irritation in rabbits.

 

In vitro eye irritation (OECD 405)

This in vitro study was performed to assess the corneal irritation and damage potential of test substance by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437 and INVITTOX protocol no. 98 under GLP. After a first opacity measurement of the fresh bovine corneae (t0), the test item test substance, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 2 °C in cMEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240). After the opacity measurements permeability of the corneae was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °C in a horizontal position. The coming out liquid was measured spectrophotometrically. With the negative control (0.9 % NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (10 % (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant.

The test item test substance did not cause any permeability of the corneae compared with the results of the negative control. Due to the dying property of the test item the corneae were coloured but still translucent after the treatment period, thus the increase of the opacity value compared with the results of the negative control is most probably caused by the dying effect. The calculated mean in vitro score was 19.34 and therefore, the test item has to be classified as mild eye irritant. But the actual effect of the present test might be weaker than the irritation score indicates. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item test substance is considered to be a mild eye irritant according to the criteria proposed in the INVITTOX protocol no. 98. Based upon the classification criteria according to „Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008“ the test item is not classified as Eye Effects Category 1 (irreversible effects, eye corrosion).

 

In vivo eye irritation (OECD 405)

The primary eye irritation potential of FAT 40851/B was investigated according to OECD 405. The test item was applied by instillation of 0.1 g into the right eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed after 1, 24, 48 and 72 hours after test item instillation. The instillation of FAT 40851/B into the eye resulted in slight early-onset and transient occular changes, such conjunctival reddening, chemosis and discharge. The mean 24/48/72 h corneal opacity, iris, conjunctivae and chemosis scores were 0, 0, 0.58 and 0.08 respectively. The conjunctivae and chemosis was reversible withing 72 h. No abnormal findings were observed in the cornea or iris of any animal at any of the examination. Based on the results and according to the Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, FAT 40851/B does not have to be classified as eye irritant.

Justification for classification or non-classification

Based on the aforementioned results, FAT40851/A needs not to be classified as skin irritant according to the criteria laid down in Regulation (EC) 1272/2008 and Directive 67/548/EEC. Based on the results of the in vivo study performed and according to the Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, FAT 40851/B does not have to be classified as eye irritant.