Bis(2-ethylhexyl) succinate

Endpoint summary

Description of key information

Studies conducted to recignised testing guidelines with GLP certification.

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:

4 February 2013 to 9 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Test system:

human skin model

Source species:

human

Cell source:

other: reconstructed human epidermal model that is grown from human-derived epidermal keratinocytes

Details on animal used as source of test system:

The model is a three-dimensional reconstructed human epidermis model consisting of human-derived epidermal keratinocytes.
Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

Pre-Test
-Assessment of Direct Test Item Reduction of MTT
In order to assess the potential non-specific reduction of the test material, 40 µL of test material was added to 0.3 mL of 1.0 mg/mL MTT and the colour change was assessed after three hours. There was no change in colour therefore the test material did not interact with MTT.

Main Test
Application of Test and Control Substances
On the day of receipt EpiDerm™ tissues were placed in a refrigerator. The next day, at least one hour before starting the assay, the tissues were transferred to 6-well plates with the assay medium, which was replaced immediately before the test was started. The test was performed on a total of four tissues per test material, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. A volume of 40mL of the undiluted test material was added into the MILLICELL insert on top of the Epi-200 tissues. Further tissues were concurrently treated with 40mL distilled water (negative control) and with 40mL 8N potassium hydroxide (positive control). After the 3-minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed.

Cell Viability Measurements Once all tissues had been rinsed, they were transferred to wells containing 300mL of 1 mg/mL MTT-medium and were incubated for 3 hours (37°C). After incubation, the tissues were washed with PBS and any resultant colour was extracted with 2 mL isopropanol overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test material was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Data Evaluation
The OD values obtained for each test sample were used to calculate the percentage viability relative to the negative control, which is arbitrarily set at 100 %. The prediction of corrosivity associated with the EpiDerm™ model is:
1. The test material is considered to be corrosive to skin if the viability after a three minute exposure is less than 50 %, or if the viability after three minutes exposure is greater than or equal to 50 % and the viability after one hour is less than 15% .
2. The test material is considered to be non-corrosive to skin if the viability after a three minute exposure is greater than or equal to 50 % and the viability after a one hour exposure is greater than or equal to 15 %.

Justification for test system used:

The test has been scientifically validated and granted regulatory approval for the identification and classification of the corrosive potential of chemicals.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

A volume of 40mL of the undiluted test material was added into the MILLICELL insert on top of the Epi-200 tissues.

Duration of treatment / exposure:

2 replicates: 3 minute exposure
2 replicates: 1 hour exposure

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2 replicates for each exposure duration (3 minute and 1 hour) and type (positive control, negative control, and treatment)

Details on test animals or test system and environmental conditions:

EpiDerm™: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek Corporation, Ashland, Massachusetts, USA.

Amount / concentration applied:

40 µL mg of the test material was applied topically.

Duration of treatment / exposure:

3 and 60 minutes.

Observation period:

3 hours

Number of animals:

Not applicable

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

3 minute exposure

Value:

13.6

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

Remarks:

mean

Run / experiment:

1 hour exposure

Value:

16.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: OD570

Run / experiment:

Mean / 1 hour exposure

Value:

1.67

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Max score 1.821

Skin viability after a three minute or one hour exposure to the test material was 93 % and 89 %, respectively.

Skin viability after a three minute or one hour exposure to the positive control article was 18 % and 11 %, respectively, demonstrating appropriate performance of the assay.

Table 1: Three Minute Exposure Period

Substance	OD570			Mean	Tissue mean	% viability	% survival
Negative	2.007	2.015	2.041	2.021	1.854	16.5	100
Negative	1.669	1.686	1.705	1.687
Test material	1.597	1.593	1.625	1.605	1.732	13.6	93
Test material	1.0853	1.862	1.860	1.859
Positive	0.354	0.342	0.361	0.352	0.330	12.7	18
Positive	0.307	0.304	0.311	0.307

 

 

Table 2: One Hour Exposure Period

Substance	OD570			Mean	Tissue mean	% viability	% survival
Negative	1.957	1.924	1.923	1.935	1.879	5.7	100
Negative	1.821	1.794	1.857	1.824
Test material	1.525	1.511	1.519	1.518	1.670	16.6	89
Test material	1.827	1.810	1.826	1.821
Positive	0.177	0.184	0.174	0.178	0.200	19.3	11
Positive	0.225	0.209	0.230	0.221

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study, the test material was considered not to be corrosive to the skin in the in vitro skin model EpiDerm™.

Executive summary:

The potential of the test material to be corrosive to skin was investigated in vitro, in a GLP study which was conducted in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis.

During the study, duplicate

EpiDerm™ inserts were treated with test material, distilled water (negative control) and 8N potassium hydroxide (positive control) for 3 minutes and 1 hour. At the end of the treatment period, the tissues were washed with phosphate buffered saline and cell viability was assessed using the MTT assay. The skin corrosivity potential was classified according to the remaining cell viability obtained after test material treatment with either of the two treatment times.

Skin viability after a three minute or one hour exposure to the test material was 93 % and 89 %, respectively.

Skin viability after a three minute or one hour exposure to the positive control article was 18 % and 11 %, respectively, demonstrating appropriate performance of the assay.

Under the conditions of the study, the test material was considered not to be corrosive to the skin in the in vitro skin model EpiDerm™.

Reference 2

Endpoint:

skin irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 February 2013 to 10 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell source:

other: EpiDerm™: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek Corporation, Ashland, Massachusetts, USA.

Justification for test system used:

The test has been scientifically validated and granted regulatory approval for the identification and classification of the corrosive potential of chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

The model is a three-dimensional reconstructed human epidermis model consisting of human-derived epidermal keratinocytes.
Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

Pre-Test
-Assessment of Direct Test Item Reduction of MTT
In order to assess the potential non-specific reduction of the test material, 30 µL of test material was added to 0.3 mL of 1.0 mg/mL MTT and the colour change was assessed after three hours. There was no change in colour therefore the test material did not interact with MTT.

- Assessment of staining potential for non-coloured substances
In order to assess the potential of staining, 30 µL of the test material was added to 0.3 mL deionised water in a suitable glass container and incubated for 60 minutes at 37 ± 1ºC, 5 ± 1% CO2. At the end of the incubation, the mixture was shaken. No staining was recorded and the test material was deemed not to have the potential to stain the tissue.

- Assessment of mesh compatibility To prevent capillary effects drawing the liquid test material to the edge of the insert a nylon mesh was recommended. An assessment of the interaction of the mesh and test material was therefore performed. A nylon mesh was placed onto a glass microscope slide and 30 µL of the test material was applied. After exposure for 60 minutes, the mesh was examined microscopically. No signs of interaction were noted therefore the test material was applied using the mesh as a spreading aid.

Main Test
- Application of test and control substances
EpiSkin™ tissues were kept in their packaging at room temperature in a microbiological safety cabinet until the next step. The tissues were set up 24 hours prior to treatment by placing each tissue onto 2 mL pre-warmed maintenance medium (supplied with the EpiSkin™ tissues) in 12-well plates and incubating at 37 °C.
The test was performed on a total of three tissues per test material, negative and positive control. Immediately prior to treatment initiation, the media under the tissues was replaced with 0.9 mL of pre-warmed assay medium (supplied with the EpiSkin™ tissues).
A volume of 30mL of the undiluted test material was added topically to the tissues. A volume of 30 mL of the positive and negative control solutions was used.
The tissues were incubated at 37 ± 1 ºC, 5 ± 1 % CO2 for 35 minutes ± 1 minute. After this period, the plates were removed from the incubator and placed into a sterile hood until the 60 ± 1 minute treatment period was complete for each tissue. Following treatment, substances were removed by washing the tissues. The tissues were then placed on the appropriate medium and incubated for 42 ± 2 hours.

- Cell viability measurements
Upon completion of the 42 hour recovery period, the base of each tissue was rinsed with Phosphate Buffered Saline (PBS) before being placed on top of 2 mL of 0.3 mg/mL (final concentration) MTT in medium and incubated for three hours (37 °C, 5 % CO2). After incubation, the tissues were washed with PBS and any resultant colour was extracted with 2 mL isopropanol, with plate shaking, for 2 hours. Upon completion of the extraction each tissue was pierced using a hypodermic needle and the extract drained and mixed well.
Two x 200 µL aliquots of each extractant were placed into a 96-well plate. The optical density of each resultant extract was determined spectrophotometrically at 570 nm, using extraction solvent as a blank and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue.

Data Evaluation
- Assay acceptance criteria
The assay was considered valid if the following criteria were met:
1. The absolute OD570 of the negative control tissues in the MTT test is an indication of the tissue viability in the testing laboratory after the shipping and storage procedure and under specific conditions of the assay. The OD values for the negative controls should be between 0.6 and 1.5, for this tissue model.
2. The viability of the tissues treated with the positive control should be ≤ 40 %.
3. The standard deviation (SD) for tissues should be less than 18 %.

- Interpretation of results
A test material that reduces the viability of the epidermal tissue to below 50 % of the negative control is predicted to be an IRRITANT (GHS category 2). A test material that does not reduce the viability of the epidermal tissue to below 50 % of the negative control is predicted to be a NON-IRRITANT (no label).

Amount/concentration applied:

30 µL mg of the test material was applied topically.

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three replicates per test material, negative and positive control.

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Run / experiment:

Negative control

Value:

100

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

Remarks:

Mean

Run / experiment:

Test material

Value:

100

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

Remarks:

Mean

Run / experiment:

Positive control

Value:

4.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Cell Viability Measurements

Substance	Tissue replicate	OD570		Corrected mean	Standard deviation	Coefficient of variance	% relative survival
		Aliquot 1	Aliquot 2				Tissue	mean
Negative control	A	1.460	1.538	1.499	7.08	7.084	108.1	100.0
	B	1.261	1.371	1.316			94.9
	C	1.309	1.382	1.345			97.0
Test material	A	1.624	1.885	1.755	15.20	13.823	126.5	110.0
	B	1.312	1.369	1.340			96.7
	C	1.318	1.640	1.479			106.7
Positive control	A	0.049	0.057	0.053	0.46	10.739	3.8	4.3
	B	0.063	0.069	0.066			4.7
	C	0.055	0.064	0.060			4.3
Blank		0.000	0.000

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test material was considered to be a 'non-irritant' to the skin.

Executive summary:

The skin irritation potential of the test material was investigated in vitro, in a GLP study which was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46.

During the study

EpiSkin™ inserts were treated with test material, negative control (phosphate buffered saline (PBS)) and positive control (5% w/v sodium dodecyl sulphate (SDS)) for 15 minutes. At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the MTT assay. The skin irritation potential was classified according to the remaining cell viability obtained after test article treatment.

The group mean viability for the test article was 110.0 %, for the negative control was 100.0 % and for the positive control was 4.3 %.

Under the conditions of the study the test material was considered to be a 'non-irritant' to the skin.

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:

21 November 2012 to 21 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Species:

cattle

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

Measurement of pH
As a preliminary test, the pH of the test material was checked. A 50 % w/v dispersion in purified waster had a pH of 4. Since this was within the acceptable range of pH 2.0 - 11.5, the study continued.

BCOP Assay
Fresh corneas, mounted onto specifically designed holders were treated topically with the test material. Eye corrosion / severe irritation potential was based on the combined effect of the test material on the opacity of the cornea following the treatment and the cornea’s ability to resist penetration of a fluorescent dye through the tissue. The assay was conducted in accordance with OECD Guidelines for Testing of Chemicals Method 437 (adopted 7 September 2009).

Irritation parameter:

cornea opacity score

Run / experiment:

Mean corrected opacity

Value:

1.7

Negative controls validity:

valid

Remarks:

Mean corrected capacity = 0

Positive controls validity:

valid

Remarks:

Mean corrected capacity = 50

Irritation parameter:

other: Corneal Permeability

Run / experiment:

Mean group Corrected OD490

Value:

-0.024

Negative controls validity:

valid

Remarks:

Mean group Corrected OD490 = 0

Positive controls validity:

valid

Remarks:

Mean group Corrected OD490 = 0.217

Irritation parameter:

in vitro irritation score

Run / experiment:

IVIS (mean opacity + (15 x mean permeability))

Value:

1.31

Negative controls validity:

valid

Remarks:

IVIS = 0

Positive controls validity:

valid

Remarks:

IVIS = 53.26

Other effects / acceptance of results:

The test material produced an IVIS score of 1.31 and was considered not to be corrosive or severely irritating to the eye.

The results for the negative and positive control articles met the acceptance criteria specified in the protocol.

Table 1: Corneal Opacity

Test substance	Cornea number	Initial opacity	Post incubation opacity	Change in opacity	Mean change in opacity	Corrected opacity	Mean corrected opacity
Test material	35	5	8	3	N/A	1.3	1.7
	37	3	6	3		1.3
	39	2	6	4		2.3
Negative control	13	3	3	0	1.67	-1.7	0
	15	3	6	3		1.3
	36	4	6	2		0.3
Positive control	8	2	60	58	N/A	56.3	50
	16	3	61	58		56.3
	17	3	42	39		37.3

Table 2: Corneal Permeability

Test substance	Cornea number	Mean blank OD490	OD490	Corrected OD490	Mean Corrected OD490	Final Corrected OD490	Mean group Corrected OD490
Test material	35	-	0.035	0.035	N/A	-0.018	-0.024
	37		0.011	0.011		-0.043
	39		0.043	0.043		-0.01
Negative control	13	0	0.062	0.062	0.053	0.009	0
	15		0.041	0.041		-0.012
	36		0.056	0.056		0.003
Positive control	8	-	0.304	0.304	N/A	0.251	0.217
	16		0.298	0.298		0.245
	17		0.21	0.21		0.157

Table 3: Calculated IVIS

Test substance	Mean opacity	Mean permeability	IVIS (mean opacity + (15 x mean permeability))
Negative control	0	0	0
Positive control	50	0.217	53.26
Test article	1.7	-0.024	1.31

The test material produced an IVIS score of 1.31 and was considered not to be corrosive or severely irritating to the eye.

The results for the negative and positive control articles met the acceptance criteria specified in the protocol.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study, the test material was considered not to be corrosive or severely irritating to the eye.

Executive summary:

The purpose of the study was to determine the eye corrosion potential of the test material following the standardised guideline OECD 437. The study was conducted under GLP conditions. Bovine corneas were used and the effect of the test substance on opacicity and permeability was examined.

Under the conditions of the study the test material was considered not to be corrosive or severely irritating to the eye. The in vivo test was therefore conducted.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2012 to 21 December 2012

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:

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
- Age at study initiation: 13 - 14 weeks (on day 1)
- Weight at study initiation: 2.7 - 3.0 kg (on day -1)
- Housing: individually housed in cages that conformed to the 'Code of Practice for Housing and Care of Animals Used in Scientific Procedures' (Home Office, London, UK)
- Diet: ad libitum
- Water: mains water, ad libitum
- Acclimation period: 21 - 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 22 °C
- Humidity (%): not less than 43 %
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 10 December 2012 To: 21 December 2012

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

- Dose administration: 0.1 mL of the test material was placed into the left conjunctival sac of one rabbit, by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were then held together for a few seconds to prevent loss of the test material. The right eye remained untreated and was used for control purposes. After consideration of the ocular responses in the first treated animal, two further animals were treated.

Observation period (in vivo):

Animals were observed up to 72 hours post administration

Number of animals or in vitro replicates:

One male initially, followed by a further two males once the irritation potential was fully assessed in the first animal.
Only rabbits with eyes free from damage or irritation were accepted onto the study.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Irrigation was not performed

SCORING SYSTEM:
The reactions observed were scored in accordance with the criteria of Draize (1977).

TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by the use of a pencil-beam torch. Furthermore, at examinations carried out 24, 48 and 72 hours after treatment the cornea was subject to application of 1 % aqueous fluorescein solution followed by irrigation with water and illumination by an uv source. Damage to the corneal epithelium was revealed by absorption of the fluorescing dye into epithelium or stroma.

Irritation parameter:

cornea opacity score

Remarks:

Mean

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

Mean

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Remarks:

Mean

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Remarks:

Mean

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

Mean

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

Mean

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Mean

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Mean

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Mean

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

Mean

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

Mean

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

Mean

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Scattered or diffuse areas of corneal opacity were noted in two treated eyes 24 and 48 hours after instillation. There were no irridial effects. Minimal conjunctival irritation was noted in all treated eyes 30 minutes, 1 hour and 4 hours after instillation. The eyes of all animals appeared normal by the 72 hour examination.

Other effects:

Instillation of the test material caused no initial sting response.

No observations indicative of systemic toxicity or ill health were noted for any rabbits during the course of the study.

Table 3: Individual Ocular Response

Rabbit number, sex , initial and final body weights	63 Male (2.7 - 2.7 kg)						64 Male (2.7 - 2.9 kg)						65 Male (3.0 – 3.1 kg)
	Initial sting response = 0						Initial sting response = 0						Initial sting response = 0
Time after treatment	½ hr	1 hr	4 hrs	24 hrs*	48 hrs	72 hrs	½ hr	1 hr	4 hrs	24 hrs*	48 hrs*	72 hrs*	½ hr	1 hr	4 hrs	24 hrs*	48 hrs*	72 hrs*
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	1	1	0	0	0	0	1	1	0
F = Area of Opacity	0	0	0	0	0	0	0	0	0	1	1	0	0	0	0	1	1	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	5	5	0	0	0	0	5	5	0
IRIS
D = Reaction	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	1	1	1	0	0	0	1	1	1	0	0	0	1	1	1	0	0	0
B = Chemosis	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
C = Discharge	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Score (A + B + C) X 2	2	2	2	0	0	0	2	2	2	0	0	0	2	2	2	0	0	0
Total Score	2	2	2	0	0	0	2	2	2	5	5	0	2	2	2	5	5	0

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, the test material only elicited slight reactions in any of the animals during the course of the study, meaning that the test material does not require classification as an eye irritant.

Executive summary:

The eye irritation potential of the test material was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 405 and EU Method B.5. Approximately 0.1 mL of test material was applied into one one of each of three rabbits and were assessed for up to 72 hours to determine the grade of ocular reaction. Instillation of the test material caused no initial sting response. Scattered or diffuse areas of corneal opacity were noted in two treated eyes 24 and 48 hours after instillation. There were no iris effects. Minimal conjunctival irritation was noted in all treated eyes 30 minutes, 1 hour and 4 hours after instillation. The eyes of all animals appeared normal by the 72 hour examination. Under the conditions of the test, the test material only elicited slight reactions in any of the animals during the course of the study that meant the test material does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

The potential of the test material to be corrosive to skin was investigated in vitro, in a GLP study which was conducted in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis. During the study, duplicate EpiDerm™inserts were treated with test material, distilled water (negative control) and 8N potassium hydroxide (positive control) for 3 minutes and 1 hour. At the end of the treatment period, the tissues were washed with phosphate buffered saline and cell viability was assessed using the MTT assay. The skin corrosivity potential was classified according to the remaining cell viability obtained after test material treatment with either of the two treatment times. Skin viability after a three minute or one hour exposure to the test material was 93 % and 89 %, respectively. Skin viability after a three minute or one hour exposure to the positive control article was 18 % and 11 %, respectively, demonstrating appropriate performance of the assay. Under the conditions of the study, the test material was considered not to be corrosive to the skin in the in vitro skin model EpiDerm™.

 

Subsequently, the skin irritation potential of the test material was investigated in vitro, in a GLP study which was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46. During the study EpiSkin™ inserts were treated with test material, negative control (phosphate buffered saline (PBS)) and positive control (5% w/v sodium dodecyl sulphate (SDS)) for 15 minutes. At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the MTT assay. The skin irritation potential was classified according to the remaining cell viability obtained after test article treatment. The group mean viability for the test article was 110.0 %, for the negative control was 100.0 % and for the positive control was 4.3 %. Under the conditions of the study the test material was considered to be a 'non-irritant' to the skin.

An in vivo skin irritation study is not required as the existing in vitro data is adequate to address this endpoint.

Eye

The eye irritation potential of the test material was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 437, OECD 405 and EU Method B.5.

A bovine corneal opacity and permeability assay (BCOP) was initially conducted. This demonstrated that the test material did not have the potential to cause corrosion or severe irritation to the eye. An in vivo test was therefore conducted. The undiluted test material (0.1 mL) was instilled into one conjunctival sac of each of three New Zealand White rabbits on Day 1. Ocular reactions were assessed for up to three days after treatment. Ocular instillation of the test article provoked no initial sting reaction. Instillation of the test material produced scattered or diffuse areas of corneal opacity and minimal conjunctival irritation. There were no iridial effects. The eyes of all rabbits were overtly normal by the 72-hour examination. Under the conditions of the test, the test material only elicited slight reactions in any of the animals during the course of the study that meant the test material does not require classification as an eye irritant.

Justification for selection of skin irritation / corrosion endpoint:

Two key studies are available to address skin irritation and corrosion.

Justification for selection of eye irritation endpoint:

Two key studies are available to address eye irritation.

Justification for classification or non-classification

Skin

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for skin irritation.

Eye

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for eye irritation.