Propionaldehyde, reaction product with formaldehyde

Endpoint summary

Description of key information

Skin irritation/Corrosion:

OECD 404, EU Method B.4: not irritating to the skin

OECD 431, in vitro: not corrosive to the skin

Eye irritation/Corrosion:

OECD 405, EU Method B.5: not irritating

OECD 437, in vitro: not corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 17 March 2010 to 18 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

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

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species and strain: Rabbits, New Zealand White.
Supplier: Charles River Deutschland GmbH, D-88353 Kisslegg.
Number and sex: 3 females.
Body weight (at the start and at the termination of the study):
Animal No. 1: 2.1 kg and 2.3 kg.
Animal No. 2: 2.5 kg and 2.5 kg.
Animal No. 3: 2.4 kg and 2.5 kg.

Hygiene: Optimal hygienic conditions.
Room number: EI1-8.
Room temperature: Ranges from 19.32 to 20.83 °C.
Relative humidity: Ranges from 44.05 to 57.72 %.
Air exchange: 12 per hour.
Light: Artificial light from 6 a.m. to 6 p.m.
Cages: Individual caging in terulan cages, Ehret GmbH, A-3430 Tulln, 65 cm x 65 cm bottom area, 50 cm height.
Feed: Ssniff K-H maintenance diet for rabbits (item V-2333-000), ad libitum, supplied by Ssniff Spezialdiäten GmbH, 59494 Soest, Germany.
Water: Tap water from an automatic watering system, ad libitum.
Identification: Labelling with felt-tipped pen on a pinna and cage labels.
Acclimatisation: 7 days (animal No. 1) and 14 days (animal Nos. 2 and 3)

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 g per animal

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48 and 72 hours after patch removal.

Number of animals:

3 females

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- Type of wrap if used: Self adhesive non woven fabric, hypoallergenic, made by Beiersdorf AG, D-20245 Hamburg.
Hair was clipped on the dorsal areas of the trunks one day before the application of the test substance. An electric hair clipper, Aesculap GH 204 with a 1 mm cutterhead, was used.
The test sites were median on the dorsal thoracal region.
Samples with approximately 0.5 g of the test substance were moistened with 1.0 mL deionised water and were placed on gauze patches1) in a size of about 2.5 cm x 2.5 cm and were applied to the test sites. They were held in place by fixing them marginally with non irritating tapes2). The application sites were covered semi-occlusively by a dressing3).
Access by the animals to the application sites was prevented by a plastic collar.

1) Pur Zellin-Tupfer, obtained by Fa. Hartmann , A-2355 Wiener Neudorf.
2) "Blenderm ®" surgical tape, hypoallergenic, 3M, Medical Products Division, St. Paul, MN 551444, USA.
3) Self adhesive non woven fabric, hypoallergenic, made by Beiersdorf AG, D-20245 Hamburg.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period the dressings, the tapes with the patches and the collars were removed.
- Time after start of exposure: The duration of the exposure was 4 hours.

OBSERVATION TIME POINTS
The skins of the animals were examined for local alterations one day before the administration of the test substance (after clipping of the hair) and immediately before the administration. The treated areas of the animals were examined for erythema/eschar and oedema as well as for other local alterations (such as hyperplasia, scaling, discolouration, fissures, scabs and alopecia) approximately 1, 24, 48 and 72 hours after patch removal.
No further examinations were performed thereafter.

SCORING SYSTEM:
- The surrounding of the administration area, i.e. the untreated skin, served as a negative control.
The skin was examined using a cold light source KL 1500 electronic.
Dermal irritation was described and recorded according to the scoring scheme for acute dermal irritation/corrosion tests.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

mean

Remarks:

all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritant / corrosive response data:

All areas treated with the test substance and all control areas were normal before the application.
Erythema/eschar: Not observed at any observation point.
Oedema: Not observed at any observation point.

Other effects:

None

 

	Mean Scores for Animal No.:
	1	2	3
Erythema / Eschar:	0.0	0.0	0.0
Oedema:	0.0	0.0	0.0

Interpretation of results:

GHS criteria not met

Remarks:

EU GHS

Conclusions:

Based on the mean scores calculated from the individual examinations performed 24, 48 and 72 h p.a. and referring to Regulation (EC) No 1272/2008 the test substance does not require classification for skin irritation.

Executive summary:

The aim of this study was to investigate possible irritation or corrosion by the test substance following a single application to the intact skin of rabbits. Methods and investigations were performed in accordance with the OECD Guideline 404 and the Council Regulation (EC) No 440/2008, Method B.4. and in accordance with GLP requirements.0.5 g test substance, moistened with deionised water, was applied via a patch to a site of 2.5 cm x 2.5 cm of the intact skin of each of 3 New Zealand White rabbits and covered by a semi-occlusive dressing. The duration of the exposure was 4 hours.

First the test substance was administered to one animal. As no serious skin reactions were noted in this animal, the remaining two animals were exposed to the test substance one week later.

Investigations on body weights were performed at the start and at the termination of the test. General signs of toxicity were checked once daily and skin examinations were done 1, 24, 48 and 72 h after patch removal (p.a.).

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Skin examinations

·      Erythema/Eschar: Not observed at any observation point.

·      Oedema: Not observed at any observation point.

The following mean scores were calculated for each animal from the examinations 24 h, 48 h and 72 h p.a.:

 

	Mean Scores for Animal No.:
	1	2	3
Erythema / Eschar	0.0	0.0	0.0
Oedema	0.0	0.0	0.0

 

 According to Regulation (EC) No 127272008 the test substance does not require classification for skin irritation.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 3 December 2009 to 11 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek´s EpiDerm System

Details on animal used as source of test system:

MatTek´s EpiDerm System consists of normal, human-derived epidermal keratinocytes which have been cultured form a multilayered, highly differentiated model of the human epidermis. It consists 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 EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm diameter) and shipped as kits, containing 24 tissues on shipping agarose.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm Skin Corrosivity Test, MatTek´s EpiDerm System
- Tissue batch number(s): EpiDerm™ tissues
- Production date: not specified
- It consists 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 EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm diameter) and shipped as kits, containing 24 tissues on shipping agarose.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation with the test chemical, the negative (deionised water) and positive control (8N KOH) for 3 minutes or for 1 hour followed by washing with PBS (Phosphate Buffered Saline), the tissues were incubated with MTT medium at 37°C and 5 % CO2. After 3 hours, the MTT medium was aspirated from all wells and the tissues were gently rinsed with PBS (2 times). For extraction, the tissues were incubated with extractant solution (isopropanol) over night without shaking.
After the extraction period, the tissues were pierced with an injection needle and the extract (now a blue formazan solution) was allowed to run into the well from which the tissue was taken. The 24-well plates were placed on a shaker for 15 minutes until the solutions were homogeneous in colour.
- Cell viability was calculated for each tissue as percent of the mean of the negative control tissues. The skin corrosivity/irritation potential of the test substance was classified according to remaining cell viability obtained after test substance treatment with either of the two exposure times.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of the test substance were dispensed directly atop the Epi-200 tissue.

NEGATIVE CONTROL - deionised water

POSITIVE CONTROL - 8N KOH

Duration of treatment / exposure:

3 minutes
1 hour

Number of replicates:

Two tissue replicates were used for each treatment (exposure time), including deionised water as negative and 8N KOH as positive control

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

85.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Epiderm Skin Corrosivity Test:
•The mean percentage viability of the treated skin discs after 3 minutes of exposure was 85.7 % which is above the threshold of 50 % for classification.
•The mean percentage viability of the treated skin discs after 1 hour of exposure was 94.0 % which is above the threshold of 15 % for classification.

Interpretation of results:

GHS criteria not met

Remarks:

EU GHS

Conclusions:

According to the results of this study the test substance the test substance is considered to be non-corrosive to skin.

Executive summary:

The EpiDerm Skin Corrosivity Test (Model EPI-200) was performed to reveal a possible corrosive potential of the test substance according to OECD Guideline 431 and under GLP.

The test substance was topically applied for 3 minutes and for 1 hour to the EpiDermal surfaces of three-dimensional human EpiDermis models, followed by immediate determination of the cytotoxic effect.

 The mean percentage viability of the treated skin discs after 3 minutes of exposure was 85.7 % which is above the threshold of 50 % for classification.The mean percentage viability of the treated skin discs after 1 hour of exposure was 94.0 % which is above the threshold of 15 % for classification.

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:

from 27 April to 28 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

7th of September 2009.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA (1996). Label Review Manual: 2nd Edition. EPA737-B-96-001. Washington, DC: U.S. Environmental Protection Agency.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN (2007). Globally Harmonized System of Classification and Labelling of Chemicals (GHS). New York & Geneva: United Nations Publications

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Slaughterhouse Fröch, Kirchenplatz 3, A-7023 Zemendorf.
- Age at study initiation: 18 – 20 month and free of macroscopically visible defects.
Corneas: Isolated corneas from the eyes of cows and bulls aged between 18 – 20 month and free of macroscopically visible defects.
Justification: According to the guidelines.
Number of corneas: Total of 9 corneas: 3 for the test substance, 3 for the negative control and 3 for the positive control.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL per cornea
- Concentration (if solution): The test substance was administered undiluted as received. No analyses of the test substance were performed.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES
3

DETAILS
Bovine corneas were isolated and mounted in cornea holders and equilibrated for one hour to achieve normal metabolic activity. After exclusion of corneas which did not achieve quality criteria, the corneas were distributed into groups (3 per group) and exposed to the test substance, the negative control and the positive control for 10 minutes. Then the substances were removed and the corneas were accurately washed. After a post exposure incubation of two hours the opacity and permeability of each cornea were recorded. Opacity was measured quantitatively with the aid of an opacitometer. Permeability was determined by the amount of sodium fluorescein dye that penetrated all cornea layers. For this purpose fluorescein solution was filled into the anterior chambers of the cornea holders followed by an incubation period of 90 minutes. The amount of sodium fluorescein that crossed into the posterior chambers was quantitatively measured with a spectrophotometer at OD490. Using opacity and permeability data an IVIS was calculated. The positive and negative control groups were simultaneously used for other, concurrently performed studies.

NEGATIVE CONTROL USED
No treatment.

POSITIVE CONTROL USED
1 % sodium hydroxide in deionised water (sterile).

APPLICATION DOSE AND EXPOSURE TIME
750 µl test substance were introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed during exposure (close-chamber method). Then the corneas were exposed in a horizontal position for 10 minutes while ensuring that the test substance adequately covered the epithelial surface. Incubation temperature, monitored with a min / max thermometer, was 29.3 °C – 33.7 °C.

POST-INCUBATION PERIOD: yes
After the exposure period substances were removed from the anterior chamber and the epithelium was washed three times with EMEM+ to determine the effectiveness of rinsing acidic or alkaline materials. cEMEM was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Both chambers were then refilled with fresh cEMEM. The corneas were incubated for additional two hours. Incubation temperature, monitored with a min / max thermometer, was 29.3 °C – 33.7 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The substances were removed and the corneas were accurately washed.
- POST-EXPOSURE INCUBATION: After a post exposure incubation of two hours the opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was determined by the amount of light transmission through the cornea. Corneal opacity was measured quantitatively in Lux with the aid of an opacitometer-kit BASF-OP2.0 (see Appendix 2), which was calibrated with a standard filter set before the corneas were measured.
Opacity values were calculated as follows:
Opacity value = ax (I0/I) + b
I = illuminance (Lux) with the cornea
I0 = illuminance (Lux) without cornea
a, b = equipment-specific variables
The standard deviation for each group was calculated as well.

- Corneal permeability:
1 mL sodium fluorescein solution was added to the anterior chamber of the cornea holder and then incubated in a horizontal position for 90 minutes. Incubation temperature, monitored with a min / max thermometer, was 29.3 °C – 33.7 °C. Passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
Permeability was determined by the amount of sodium fluorescein dye that penetrated all corneal cell layers. The amount of sodium fluorescein that crossed into the posterior chamber was quantitatively measured with the aid of a Bio-Tek EL800 microtiter plate reader at 490nm. For measurement 360 µl of cEMEM from the posterior chamber were transferred into the wells of a 96-well microtiter plate (triplicates). Data were recorded as OD490 values which were equivalent to the OD490 values based upon a visible light spectrophotometer using a standard 1 cm path length.
To proof that the measurement was performed in the linear range wells containing ten concentrations (ranging from 0.625 µg/ml to 40 µg/ml) of fluorescein solutions were additionally prepared.
To ensure that the permeability values measured were in the exponential range the linearity was determined by triple measurement of fluorescein solutions of ten concentrations. A linear calibration function and the correlation coefficient thereof was calculated.
As the OD490 values of corneas treated with 1 % sodium hydroxide lied outside of the linear range dilutions of 1:5 were measured.
The standard deviation for each group was calculated as well.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity and permeability values for each treatment group were combined in an empirically-derived formula to calculate the IVIS for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The standard deviation for each group was calculated as well.


DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used

Irritation parameter:

cornea opacity score

Run / experiment:

1_Test substance

Value:

-0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

2_Test substance

Value:

-0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

3_Test substance

Value:

-0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

1_Test material

Value:

-0.021

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

2_Test material

Value:

-0.008

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

3_Test material

Value:

0.01

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

1_Test material

Value:

-1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

2_Test material

Value:

-0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

3_Test material

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The Opacity value of the test substance was -0.6, 15 x Permeability value was -0.090 which resulted in an IVIS of -0.6.
The mean opacity and the mean permeability of the corneas, treated with deionised water (NC) were -1.0 and 0.000 respectively, which are less than the established upper limits for background opacity and permeability values of the historical data of the negative controls.
The mean IVIS of the corneas, treated with 1 % sodium hydroxide (PC) was 123.5, which is within the range of the historical data.

All untreated eyes ("control eyes") were normal at any observation time.

Table1: Opacity, permeability and IVIS values

Opacity, permeability (1 x value measured and 15 x values for IVIS calculation) and IVIS values of test substance, negative and positive controls. Individual data, means and standard deviations (SD).

Substance	Opacity			Permeability (1x)			Permeability (15x)		IVIS
	Individual	Mean	SD	Individual	Mean	SD	Mean	SD	Individual	Mean	SD
Aqua dest. (NC)	-1.2	-1.0	0.4	0.010	0.000	0.010	0.000	0.146	-1.0	-1.0	0.3
	-0.6			-0.009					-0.7
	-1.3			-0.001					-1.4
1 % Sodium hydroxide (PC)	91.3	83.9	7.9	2.347	2.642	0.272	39.63	4.082	126.5	123.5	4.2
	75.5			2.884					118.8
	84.8			2.694					125.2
Test substance	-0.8	-0.6	0.4	-0.021	-0.006	0.015	-0.090	0.231	-1.1	-0.6	0.6
	-0.7			-0.008					-0.9
	-0.1			0.010					0.0

Unforeseen events

The temperature of the incubator was 29.3 °C – 33.7 °C instead of 31 °C – 33 °C. This deviation is considered to be of no relevance for the outcome of this study.

 

Interpretation of results:

GHS criteria not met

Remarks:

EU GHS

Conclusions:

According to the results of this study and the OECD Guideline 437, the test substance is considered to be not an ocular corrosive or severe irritant.

Executive summary:

The Bovine Corneal Opacity and Permeability Study (BCOP Test Method) was performed to reveal possible ocular corrosivity and severe irritation of the test substance, according to the OECD Guideline 437 for the testing of chemicals “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants”,.

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µl of the test substance (as is) were topicallyadministered to 3 isolated bovine corneas to the epithelial surfaces for 10 minutes. After a post-incubation period of 2 hours, the final opacity was measured. Then 1 ml of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.

Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances:

• Negative control:       sterile aqua dest.
• Positive control:        1 % sodium hydroxide.

Finally the IVIS (In Vitro Irritancy Score) was calculated as follows:

IVIS = mean opacity value + (15 x mean permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS≥55.1 is defined as ocular corrosive or severe irritant.

The IVIS for the test substance was -0.6.

IVIS of the negative control was -1.0 and for the positive control 123.5, thus demonstrating the validity of the experiment.

According to the results of this study and the OECD Guideline 437, the test substance is considered to be not an ocular corrosive or severe irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April to 18 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

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

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, D-88353 Kisslegg.
- Sex: 3 females
- Age at study initiation:
- Weight at study initiation:
Animal No. 1: 1.6 kg and 1.8 kg.
Animal No. 2: 2.3 kg and 2.4 kg.
Animal No. 3: 2.2 kg and 2.3 kg
- Housing : Optimal hygienic conditions, Room EI1-8. Individual caging in terulan cages, Ehret GmbH, A-3430 Tulln, 65 cm x 65 cm bottom area, 50 cm height.
- Diet (e.g. ad libitum): Ssniff K-H maintenance diet for rabbits (item V-2333-000), ad libitum, supplied by Ssniff Spezialdiäten GmbH, 59494 Soest, Germany.
- Water (e.g. ad libitum): Tap water from an automatic watering system, ad libitum
- Acclimation period: 7 days
- Identification: Labelling with felt-tipped pen on a pinna and cage labels.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Ranges from 19.34 to 22.40 °C.
- Humidity (%): Ranges from 37.66 to 83.71 %.
- Air changes (per hr): 12 per hour.
- Photoperiod (hrs dark / hrs light): Artificial light from 6 a.m. to 6 p.m.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 mL per animal

Observation period (in vivo):

1, 24, 48 and 72 hours

Number of animals or in vitro replicates:

3 females

Details on study design:

STUDY DESIGN
0.1 mL of the test substance was administered per animal into the conjunctival sacs of the right eyes by gently pulling the lower lids away from the eyeballs for instillation. The eyes were held closed for about one second to prevent loss of test substance.
The left eyes remained untreated and served as a control.
Firstly, the test substance was administered to one animal. As no evidence for a serious damage to the eye of this animal was found during the initial 72 hour observation period (no corrosive effect), the test substance was administered to the other two animals subsequently.

Both eyes of the animals were examined within 24 hours before the instillation and approximately 1, 24, 48 and 72 hours p.a.
No further examinations were performed thereafter.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): not reported


SCORING SYSTEM:
The whole eyes, especially the corneae, the irises and the conjunctivae were examined using an otoscope lamp. Eye irritation was scored and recorded using the scheme for eye lesions.


Scoring scheme for eye lesions.
                       (Grades for ocular lesions)
CORNEA
Opacity : degree of density (area most dense area is taken for reading).
0       No ulceration or opacity.
1       Scattered or diffuse areas of opacity (except for slight dulling of normal lustre),
         details of iris clearly visible.
2       Easily discernible translucent area, details of iris slightly obscured.
3       Nacreous area, no details of iris visible, size of pupil barely discernible.
4       Opaque cornea, iris not discernible through the opacity.
 
IRIS
0       Normal.
1       Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia
         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 of all these or all together).
 
CONJUNCTIVAE
Redness:(refers to the most severe effect of palpebral and bulbar conjunctivae in comparison to the control eye).
 
0       Blood vessels normal.
1       Some blood vessels definitely hyperaemic (injected).
2       Diffuse crimson colour, individual vessels not easily discernible.
3       Diffuse beefy red.
 
Chemosis:lids and/or nictating membranes.
0       No swelling.
1       Any swelling above normal (includes nictating membranes).
2       Obvious swelling with partial eversion of lids.
3       Swelling with lids about half closed.
4         Swelling with lids more than half closed.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Irritant / corrosive response data:

All eyes, treated with the test substance ("test eyes") were normal before the instillation and at each observation time.
All untreated eyes ("control eyes") were normal at any observation time.

Other effects:

None

 

	Mean Scores for Animal No.:
	1	2	3
Cornea:	0.0	0.0	0.0
Iris:	0.0	0.0	0.0
Conjunctivae, redness:	0.0	0.0	0.0
Conjunctivae, chemosis:	0.0	0.0	0.0

Individual data

 Examination of corneae, irises and conjunctivae. Individual data and means.
              

							Conjunctivae
Time after instillation (p.a.)	Corneae animal No.			Irises animal No.			Redness animal No.			Chemosis animal No.
	1	2	3	1	2	3	1	2	3	1	2	3
1 h	0	0	0	0	0	0	0	0	0	0	0	0
24 h	0	0	0	0	0	0	0	0	0	0	0	0
48 h	0	0	0	0	0	0	0	0	0	0	0	0
72 h	0	0	0	0	0	0	0	0	0	0	0	0
mean (24-72 h)	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0

Interpretation of results:

GHS criteria not met

Remarks:

EU GHS

Conclusions:

The test substance was not irritating to the rabbit eye under the conditions of this test.

Executive summary:

The aim of this study was to investigate possible irritating or corrosive effects of the test substance following a single administration into the conjunctival sac of one eye of rabbits. Methods and investigations were performed in accordance with the OECD Guideline 405 and the Council Regulation (EC) No 440/2008, Method B.5 and under GLP. 0.1 mL of the test substance was instilled into the conjunctival sac of one eye of each of 3 New Zealand White rabbits. Firstly the test substance was administered to one animal. As there was no corrosive or severe irritant effect observed, the test substance was administered to two additional animals one week later.   Body weights were investigated at the start and at the termination of the test.   General signs of toxicity were checked once daily. Eye examinations take place at 1, 24, 48 and 72 h after the administration (p.a.) of the test substance.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Corneae and irises were not affected at any observation point. Conjunctivae were not affected at any observation point.  

Mean scores of 0 were calculated for each animal from the individual examinations performed 24 h, 48 h and 72 h p.a for each endpoint Cornea, Iris, Conjunctivae, redness, Conjunctivae, chemosis, respectively.

Based on the mean scores calculated from the individual examinations performed 24, 48 and 72 h p.a. and referring to Regulation (EC) No 1272/2008 the test substance does not require classification for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The aim of this study was to investigate possible irritation or corrosion by the test substance following a single application to the intact skin of rabbits. Methods and investigations were performed in accordance with the OECD Guideline 404 and the Council Regulation (EC) No 440/2008, Method B.4. and in accordance with GLP requirements.0.5 g test substance, moistened with deionised water, was applied via a patch to a site of 2.5 cm x 2.5 cm of the intact skin of each of 3 New Zealand White rabbits and covered by a semi-occlusive dressing. The duration of the exposure was 4 hours.

First the test substance was administered to one animal. As no serious skin reactions were noted in this animal, the remaining two animals were exposed to the test substance one week later.

Investigations on body weights were performed at the start and at the termination of the test. General signs of toxicity were checked once daily and skin examinations were done 1, 24, 48 and 72 h after patch removal (p.a.).

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Skin examinations

·      Erythema/Eschar: Not observed at any observation point.

·      Oedema: Not observed at any observation point.

The following mean scores were calculated for each animal from the examinations 24 h, 48 h and 72 h p.a.:

 

	Mean Scores for Animal No.:
	1	2	3
Erythema / Eschar	0.0	0.0	0.0
Oedema	0.0	0.0	0.0

 

 According to Regulation (EC) No 127272008 the test substance does not require classification for skin irritation.

Skin corrosion

The EpiDerm Skin Corrosivity Test (Model EPI-200) was performed to reveal a possible corrosive potential of the test substance according to OECD Guideline 431 and under GLP.

The test substance was topically applied for 3 minutes and for 1 hour to the EpiDermal surfaces ofthree-dimensional human EpiDermis models, followed by immediate determination of the cytotoxic effect.

 The mean percentage viability of the treated skin discs after 3 minutes of exposure was 85.7 % which is above the threshold of 50 % for classification.The mean percentage viability of the treated skin discs after 1 hour of exposure was 94.0 % which is above the threshold of 15 % for classification.

Eye corrosion

The Bovine Corneal Opacity and Permeability Study (BCOP Test Method) was performed to reveal possible ocular corrosivity and severe irritation of the test substance according to the OECD Guideline 437 for the testing of chemicals “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants”,.

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µl of the test substance (as is) were topicallyadministered to 3 isolated bovine corneas to the epithelial surfaces for 10 minutes. After a post-incubation period of 2 hours, the final opacity was measured. Then 1 ml of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.

Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances:

• Negative control:       sterile aqua dest.
• Positive control:        1 % sodium hydroxide.

Finally the IVIS (In Vitro Irritancy Score) was calculated as follows:

IVIS = mean opacity value + (15 x mean permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS≥55.1 is defined as ocular corrosive or severe irritant.

The IVIS for the test substance was -0.6.

IVIS of the negative control was -1.0 and for the positive control 123.5, thus demonstrating the validity of the experiment.

According to the results of this study and the OECD Guideline 437, the test substance
the test substance is considered to be not an ocular corrosive or severe irritant.

Eye irritation

The aim of this study was to investigate possible irritating or corrosive effects of the test substance following a single administration into the conjunctival sac of one eye of rabbits.Methods and investigations were performed in accordance with the OECD Guideline 405 and the Council Regulation (EC) No 440/2008, Method B.5 and under GLP.0.1 mL of the test substance was instilled into the conjunctival sac of one eye of each of 3 New Zealand White rabbits. Firstly the test substance was administered to one animal. As there was no corrosive or severe irritant effect observed, the test substance was administered to two additional animals one week later.    Body weights were investigated at the start and at the termination of the test.  General signs of toxicity were checked once daily.Eye examinations take place at 1, 24, 48 and 72 h after the administration (p.a.) of the test substance.

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Corneae and irises were not affected at any observation point. Conjunctivae were not affected at any observation point. 

Mean scores of 0 were calculated for each animal from the individual examinations performed 24 h, 48 h and 72 h p.a for each endpoint Cornea, Iris, Conjunctivae, redness, Conjunctivae, chemosis, respectively.

Based on the mean scores calculated from the individual examinations performed 24, 48 and 72 h p.a. and referring to Regulation (EC) No 1272/2008 the test substance does not require classification for eye irritation.

Justification for classification or non-classification

No classification is derived according to Regulation (EC) No 1272/2008 as there were no adverse effects with regard to skin or eye irritation/corrosion noted in the animals in in vivo and in vitro guideline studies.