Methyl 3-pyridyl ketone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-02 - 2015-11-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP OECD 437 guideline study without deviations on the registered substance itself.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline No. 437, edition adopted 26. Jul. 2013: “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"

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

EU Method B. 47: “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” adopted 09. Dec. 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

3-Acetylpyridine

IUPAC Name:

3-Acetylpyridine

Constituent 2

Reference substance name:

Methyl 3-pyridyl ketone

EC Number:

206-496-7

EC Name:

Methyl 3-pyridyl ketone

Cas Number:

350-03-8

Molecular formula:

C7H7NO

IUPAC Name:

1-(pyridin-3-yl)ethan-1-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 3-Acetylpyridine
- Substance type: pure substance
- Storage condition of test material: Room temperature (20 ± 5 °C)
- Homogeneity: homogeneous

Test animals / tissue source

Species:

other: Bos primigenius Taurus (fresh bovine corneas)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: 0.9% NaCl in demin. water (negative control), Dimethylformamide undiluted (positive control)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

10 min

Observation period (in vivo):

2 h + 90 min post-incubation

Number of animals or in vitro replicates:

3 replicate corneas for each treatment group (negative control solution, test item and positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red
- Time after start of exposure: 10 min

SCORING SYSTEM: Opacity value, calculated from the measured absorbance at 570 nm

TOOL USED TO ASSESS SCORE: fluorescein absorbance measurement

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

other: cornea #1

Time point:

other: 10 min incubation, 210 min postincubation

Score:

58.02

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 1st experiment; IVIS>55, Eye damage Category I

Results 2

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

other: cornea #2

Time point:

other: 10 min incubation, 210 min postincubation

Score:

56.98

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 1st experiment; IVIS>55, Eye damage Category I

Results 3

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

other: cornea #3

Time point:

other: 10 min incubation, 210 min postincubation

Score:

40.21

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 1st experiment; IVIS > 3 and ≤ 55, No prediction can be made

Results 4

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

mean

Remarks:

all corneas

Time point:

other: 10 min incubation, 210 min postincubation

Score:

51.74

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 1st experiment; IVIS > 3 and ≤ 55, No prediction can be made

Results 5

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

mean

Remarks:

all corneas

Time point:

other: 10 min incubation, 210 min postincubation

Score:

86.24

Reversibility:

other: not applicable

Remarks on result:

other: Positive control; 1st experiment; IVIS>55, Eye damage Category I

Results 6

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

mean

Remarks:

all corneas

Time point:

other: 10 min incubation, 210 min postincubation

Score:

1.64

Reversibility:

other: not applicable

Remarks on result:

other: Negative control; 1st experiment; IVIS ≤ 3, No category

Results 7

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

other: cornea #1

Time point:

other: 10 min incubation, 210 min postincubation

Score:

51.85

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 2nd experiment; IVIS > 3 and ≤ 55, No prediction can be made

Results 8

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

other: cornea #2

Time point:

other: 10 min incubation, 210 min postincubation

Score:

34.5

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 2nd experiment; IVIS > 3 and ≤ 55, No prediction can be made

Results 9

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

other: cornea #3

Time point:

other: 10 min incubation, 210 min postincubation

Score:

40.14

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 2nd experiment; IVIS > 3 and ≤ 55, No prediction can be made

Results 10

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

mean

Remarks:

all corneas

Time point:

other: 10 min incubation, 210 min postincubation

Score:

42.16

Reversibility:

other: not applicable

Remarks on result:

other: Test item; 2nd experiment; IVIS > 3 and ≤ 55, No prediction can be made

Results 11

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

mean

Remarks:

all corneas

Time point:

other: 10 min incubation, 210 min postincubation

Score:

28.53

Reversibility:

other: not applicable

Remarks on result:

other: Positive control; 2nd experiment; IVIS > 3 and ≤ 55, No prediction can be made

Results 12

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

mean

Remarks:

all corneas

Time point:

other: 10 min incubation, 210 min postincubation

Score:

0.27

Reversibility:

other: not applicable

Remarks on result:

other: Negative control; 2nd experiment; IVIS ≤ 3, No category

Irritant / corrosive response data:

In the negative control, no signs of eye irritation were observed in both experiments.
In the first experiment, the positive control induced serious eye damage, which would be classified as GHS category 1. In the second experiment, the positive control showed effects on the cornea of the bovine eye.
The test item 3-Acetylpyridine showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 51.74 in the first experiment and 42.16 in the second experiment.

Any other information on results incl. tables

Findings and Results

Two experiments were performed. The first experiment was insufficient for classification because 2 of the 3 replicates gave discordant predictions from the mean. The second experiment was valid and sufficient for classification.

 

Opacity and Permeability Values of the First Experiment

For each treatment group (negative control solution, test item and positive control), three replicates were measured.

The absorbance (570 nm) and opacity values which were measured before and after exposition are given in the following table:

 

Table Absorbance and Opacity Values Negative Control

Parameter	Negative Control
Absorbance before exposition	0.1470	0.1538	0.1572
Absorbance after exposition	0.1946	0.1689	0.2103
Opacity before exposition	1.4028	1.4250	1.4362
Opacity after exposition	1.5653	1.4754	1.6229
Opacity Difference	0.1625	0.0504	0.1868

Mean opacity difference of the negative control is 0.1332.

 

Table Absorbance and Opacity Values Test Item and Positive Control

Parameter	Test Item3-Acetylpyridine			Positive Control
Absorbance before exposition	0.1266	0.1427	0.1681	0.1947	0.1535	0.1125
Absorbance after exposition	1.4215	1.4031	1.4020	1.9553	1.4636	2.0453
Opacity before exposition	1.3384	1.3890	1.4727	1.5657	1.4240	1.2957
Opacity after exposition	26.3937	25.2988	25.2348	90.2194	29.0804	110.9941
Opacity Difference	25.0552	23.9098	23.7622	88.6537	27.6564	109.6984

 

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

 

Table Optical density at 490 nm

Replicate	Negative Control			Test Item 3-Acetylpyridine			Positive Control
Measured values	0.0181	0.0290	0.0133	0.4614	0.4629	0.2412	0.0873	0.2001	0.2144
*Corrected values	0.0905	0.1450	0.0665	2.3070	2.3145	1.2060	0.4365	1.0005	1.0720
Mean	0.1007			--

*Note: In order to correct the path length, a factor of 5 was taken into account when calculating the IVIS.

 

IVIS Values of the First Experiment

IVIS was calculated using the values in tables and the equation stated above.

Example:

IVIS (Test Item 3-Acetylpyridine, Repl. 1) = (25.0552– 0.1332) + [15 * (2.3070– 0.1007)] = 58.02

 

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

 

Table IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0.9% NaCl	1.52	1.64	32.3%
	2.23
	1.18
Test Item 3-Acetylpyridine	58.02	51.74	19.3%
	56.98
	40.21
Positive Control DMF undiluted	93.56	86.24	48.7%
	41.02
	124.14

 

 

Opacity and Permeability Values of the Second Experiment

For each treatment group (negative control solution, test item and positive control), three replicates were measured.

The absorbance (570 nm) and opacity values which were measured before and after exposition are given in the following table:

 

Table Absorbance and Opacity Values Negative Control

Parameter	Negative Control
Absorbance before exposition	0.1510	0.1524	0.1513
Absorbance after exposition	0.2742	0.2410	0.2125
Opacity before exposition	1.4158	1.4204	1.4168
Opacity after exposition	1.8802	1.7418	1.6312
Opacity Difference	0.4644	0.3214	0.2144

Mean opacity difference of the negative control is 0.3334.

 

Table Absorbance and Opacity Values Test Item and Positive Control

Parameter	Test Item3-Acetylpyridine			Positive Control
Absorbance before exposition	0.1718	0.1233	0.1739	0.1920	0.1791	0.2384
Absorbance after exposition	1.3855	1.2214	1.3230	1.4546	1.2253	1.4819
Opacity before exposition	1.4853	1.3283	1.4925	1.5560	1.5104	1.7314
Opacity after exposition	24.2941	16.6495	21.0378	28.4839	16.7996	30.3319
Opacity Difference	22.8088	15.3211	19.5453	26.9280	15.2892	28.6005

 

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

 

Table Optical density at 490 nm

Replicate	Negative Control			Test Item3-Acetylpyridine			Positive Control
Measured values	-0.0009	-0.0013	-0.0005	0.3908	0.2593	0.2781	0.0695	0.0689	0.0693
*Corrected values	-0.0045	-0.0065	-0.0025	1.9540	1.2965	1.3905	0.3475	0.3445	0.3465
Mean	-0.0045			--

*Note: In order to correct the path length, a factor of 5 was taken into account when calculating the IVIS.

 

IVIS Values of the Second Experiment

IVIS was calculated using the values in tables and the equation stated above.

Example:

IVIS (Test Item3-Acetylpyridine, Repl. 1) = (22.8088–0.3334) + [15 * (1.9540 – (-0.0045))] = 51.85

 

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

 

Table IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0.9% NaCl	0.40	0.27	43.6%
	0.22
	0.18
Test Item 3-Acetylpyridine	51.85	42.16	21.0%
	34.50
	40.14
Positive Control DMF undiluted	31.88	28.53	25.5%
	20.19
	33.53

 

 

Validity

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.

The negative control has to show an IVIS ≤ 3.

The validity criteria and findings are given in the following table:

 

Table Validity

Parameter	Criterion	Found	Assessment
IVIS of negative control 0.9% NaCl	≤ 3	1.64 (first experiment) 0.27 (second experiment)	ok
IVIS of positive control DMF undiluted	22.80 – 123.60	86.24 (first experiment) 28.53 (second experiment)	ok

 

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

 

Assessment

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category.

 

Table Classification Scheme

IVIS	UN GHS Category Eye Damage
≤ 3	No category
> 3 and ≤ 55	No prediction can be made
> 55	Eye damage Category I

 

In the negative control, no signs of eye irritation were observed in both experiments.

In the first experiment, the positive control induced serious eye damage, which would be classified as GHS category 1. In the second experiment, the positive control showed effects on the cornea of the bovine eye.

The test item 3-Acetylpyridine showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 51.74 in the first experiment and 42.16 in the second experiment.

Applicant's summary and conclusion

Interpretation of results:

other: inconclusive, no prediction can be made

Conclusions:

Testing was performed via a GLP OECD 437 guideline study on the registered substance itself. The present in vitro method is recommended in a tiered testing approach and the validity criteria are met, making the results sufficiently reliable to assess the corrosivity of 3-Acetylpyridine to the eye. The present in vitro method allows the identification of corrosive chemical substances and mixtures or of substances which do not require classification.
Two experiments were performed. The first experiment was insufficient for classification because 2 of the 3 replicates gave discordant predictions from the mean. The second experiment was valid and sufficient for classification. Under the conditions of this study, the test item 3-Acetylpyridine showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 51.74 in the first experiment and 42.16 in the second experiment.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage.
Hence, no conclusion can be drawn whether the substance needs to be classified as eye irritant, for a clear distinction an in vivo study would be required based on the current state of the art. However, based on the tonnage band o the substance, in vivo testing is not required, and with regard to animal welfare no further testing will be performed and the result of the present study will be considered inconclusive.

Executive summary:

In an OECD 437 (in vitro eye corrosion) study under GLP, two experiments were performed. The first experiment was insufficient for classification because 2 of the 3 replicates gave discordant predictions from the mean. The second experiment was valid and sufficient for classification.

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old.

The test item 3-Acetylpyridine was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 h and whose opacity had been measured. The test item was incubated on the cornea for 10 min. at 32 ± 1 °C. After removal of the test item and 2 h post-incubation, opacity and permeability values were measured.

 

Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea in both experiments. The calculated IVIS (in vitro irritancy score) is 1.64 in the first experiment and 0.27 in the second experiment.

Dimethylformamide (DMF) undiluted was used as positive control. In the first experiment, the positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 86.24. In the second experiment, the positive control induced effects on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 28.53.

 

Under the conditions of this study, the test item 3-Acetylpyridine showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 51.74 in the first experiment and 42.16 in the second experiment.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage.