Butane-1,2-diol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May – 31 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

updated 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Butane-1,2-diol

EC Number:

209-527-2

EC Name:

Butane-1,2-diol

Cas Number:

584-03-2

Molecular formula:

C4H10O2

IUPAC Name:

butane-1,2-diol

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle obtained from a local abattoire as a by-product from freshly slaughtered animals
- Characteristics of donor animals: 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue: Hanks´ Balanced Salt Solution (HBSS) on ice with 100 IU/mL penicillin and 100 µg/mL streptomycin.
- Time interval prior to initiating testing: 24 h
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: 100 IU/mL penicillin and 100 µg/mL streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Test medium and temperature conditions used in the cornea holder: Eagle´s Minimum Essential Medium (EMEM) without phenol red and plugged
- Equilibration time: 70 min at 32±1 °C
- Quality check of the equilibrated corneas: macroscopic defects

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied in the test: 750 µL

NEGATIVE CONTROL
- Substance: Sodium chloride
- Concentration: 0.9% NaCl solution in deionised water
- Amount applied in the test: 750 µL
- Lot/batch no.: 3012488
- Purity: 0.9%

POSITIVE CONTROL
- Substance: Ethanol
- Amount applied in the test: 750 µL
- Lot/batch no.: STBD7546V
- Purity: > 99.8 %

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 min at 32 ± 1 °C

Number of animals or in vitro replicates:

number of eyes for the test item: 3
number of eyes for the negative control: 3
number of eyes for the positive control: 3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Test medium and temperature conditions used in the cornea holder: Eagle´s Minimum Essential Medium (EMEM) without phenol red and plugged
- Equilibration time: 70 min at 32±1 °C

QUALITY CHECK OF THE ISOLATED CORNEAS
Macroscopic defects

NUMBER OF REPLICATES
3 replicates

NEGATIVE CONTROL USED
sodium chloride 0.9% w/v

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of the test item or control items were applied to the appropriate corneas.
Incubation at 32 ± 1 ºC for 10 minutes.

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: three times
- POST-EXPOSURE INCUBATION: 32 ± 1 °C for 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD492)
- Histopathology: Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM:
- Calculation of opacity values:
The opacity value was calculated according to the following steps:
i)              opacity change= final opacity – initial opacity
ii)             corrected opacity change= opacity change – mean opacity change of the negative control
iii)            mean opacity value= mean of all corrected opacity changes per group
 
- Calculation of OD492 (optical density at 492 nm) values:
The permeability value was calculated according to the following steps:
i)              OD492 change= OD492 – mean blank OD492ii)            corrected OD492 change= OD492 change – mean OD492 change of negative control
ii)           mean OD492 value= mean of all corrected OD492 changes per group
 
- Calculation of the IVIS (in-vitro irritancy score) values:
The In-Vitro Irritancy Score (IVIS) was calculated as follows:
 IVIS = mean opacity value+ (15 x mean permeability OD492 value)
 
DECISION CRITERIA:
- Evaluation criteria:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.

- Acceptance criteria:
OECD 437: “A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently (i.e., less than once a month). The negative or solvent/vehicle control responses should result in opacity and permeability value that less than the establisher upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.”

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean out of all 3 eyes

Value:

39.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
The corneas treated with the test item were cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of 1.3 (≤ 3.0) and permeability of 0.014 (≤ 0.077). The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score of 48.6 was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post Incubation - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	6	6	8	2		0.025
	2	4	4	6	2		0.011
	3	5	4	5	0		0.005
					1.3*		0.014**		1.5
Positive Control	4	5	31	33	28	26.7	1.510	1.496
	5	4	41	42	38	36.7	0.946	0.932
	6	4	34	37	33	31.7	0.978	0.964
						31.7***		1.131***	48.6
Test Item	7	5	32	44	39	37.7	0.450	0.436
	8	3	26	37	34	32.7	0.471	0.457
	9	4	27	35	31	29.7	0.414	0.400
						33.3***		0.431***	39.8

OD = Optical density

* = Mean of the post-incubation

** = Mean permeability

*** = Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

other: Non-corrosive (Eye Irrit. 2 or not classified) according to Regulation (EC) No 1272/2008

Conclusions:

CLP: Non-corrosive (Eye Irrit. 2 or not classified) according to Regulation (EC) No 1272/2008