Benzo[b]thiophene, 7-chloro-3-methyl-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 November 2014 - 24 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 438. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

7-chloro-3-methyl-1-benzothiophene

EC Number:

605-751-3

Cas Number:

17514-68-0

Molecular formula:

C9H7ClS

IUPAC Name:

7-chloro-3-methyl-1-benzothiophene

Details on test material:

- Name of test material (as cited in study report): 7-chloro-3-methylbenzo[b]thiophene
- Physical state: liquid
- Analytical purity: 98.5%
- Lot/batch No.: I14121A
- Expiration date of the lot/batch: May, 2016
- Storage condition of test material: room temperature: 15-3ºC.

Test animals / tissue source

Species:

other: chicken.

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Age at study initiation: 7-week-old
- Weight at study initiation: 1.5 to 2.5 kg

BIOLOGICAL MATERIAL
After sedation of the chickens by electric shock and incision of the neck for bleeding, their heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container. The time interval between the collection of the chickens' heads and the use of their eyeballs in the ICE test was 30 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL

Duration of treatment / exposure:

10 seconds.

Observation period (in vivo):

4 hours after post-treatment rinse.

Number of animals or in vitro replicates:

9 eyeballs: 3 eyeballs per group (test item, positive control and negative control).

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes (physiological salt)
- Time after start of exposure: 10 seconds.

MEASURED PARAMETERS: the corneas treated with the test item and the control items were evaluated pretreatment and starting at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation times points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once (30 minutes after the end of the exposure).

SCORING SYSTEM:

Fluorescein retention:
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

Corneal opacity:
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible


Corneal swelling:
The degree of corneal swelling was determined by measuring corneal thickness using an SP-100 pachymeter (TOMEY).

Gross evaluation:
To determine whether any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the cornea were visible.

Histopathological evaluation:
Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4% solution of formaldehyde. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using Hematoxylin and Eosin. The following layers of the cornea were evaluated: anterior epithelium, anterior elastic lamina (Bowman’s membrane), corneal stroma, posterior elastic lamina (Descemet’s membrane), and posterior epithelium. All treated eyeballs were subject to this evaluation.

Results and discussion

In vivo

Irritant / corrosive response data:

On the grounds of the study results and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 3xII.

Any other information on results incl. tables

Table 1. Fluorescein retention.

Observation after time t (minutes)	Test item			Positive control (10% acetic acid)			Negative control (physiological saline)
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	1	1	1	3	3	3	0	0	0

Table 2. Corneal opacity.

Observation after time t (minutes)	Test item			Positive control (10% acetic acid)			Negative control (physiological saline)
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	1	1	1	3	3	3	0	0	0
75	1	1	1	3	3	3	0	0	0
120	1	1	1	3	3	3	0	0	0
180	1	1	1	3	3	3	0	0	0
240	1	1	1	3	3	3	0	0	0

Table 3. Corneal swelling (%).

Observation after time t (minutes)	Test item			Positive control (10% acetic acid)			Negative control (physiological saline)
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
0	-	-	-	-	-	-	-	-	-
30	2.5	2.1	1.4	8.6	15.8	10.7	-1.4	-1.0	0.0
75	4.6	4.9	2.0	18.9	23.6	20.0	-2.7	-3.4	-5.0
120	8.4	5.9	3.4	28.2	33.5	29.3	-4.7	-4.7	-6.4
180	9.1	6.9	3.7	31.3	39.8	32.4	-6.1	-5.7	-6.4
240	9.1	8.0	3.7	36.4	41.2	36.9	-10.2	-6.0	-8.4

Table 4. Gross evaluation of the treated corneas.

Observation after time t (minutes)	Test item			Positive control (10% acetic acid)			Negative control (physiological saline)
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
30	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
75	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
120	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
180	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
240	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC

NC = no changes.

SIGNS = roughening of the corneal surface.

Table 5. Evaluation of fluorescein retention.

Observation after time t (minutes)	Test item		Positive control (10% acetic acid)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	1.0	II	3.0	IV	0.0	I

Table 6. Evaluation of corneal opacity.

Observation after time t (minutes)	Test item		Positive control (10% acetic acid)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	1.0	II	3.0	IV	0.0	I
75	1.0	II	3.0	IV	0.0	I
120	1.0	II	3.0	IV	0.0	I
180	1.0	II	3.0	IV	0.0	I
240	1.0	II	3.0	IV	0.0	I

Table 7. Evaluation of corneal swelling (%).

Observation after time t (minutes)	Test item		Positive control (10% acetic acid)		Negative control (physiological saline)
	Average	ICE class	Average	ICE class	Average	ICE class
30	2.0	I	11.7	II	0.8*	I
75	3.8	I	20.8	III	3.7*	I
120	5.9	II	30.3	III	5.3*	I
180	6.6	II	34.5	IV	6.1*	I
240	6.9	II	38.2	IV	8.2*	I

* - percentage of corneal thickness decrease, no swelling

Histopathological evaluation of the cornea:

The negative control corneas had a normal histological structure.

Histopathological examinations of the positive control corneas revealed coagulation of the corneal epithelium (eyeballs no. 4 and no. 6); detachment of the posterior corneal epithelium (eyeballs no. 4, no. 5, and no. 6); dissection of the corneal stroma (eyeball no. 4). These changes confirmed corrosive properties of 10% acetic acid.

Histopathological examinations of the corneas treated with the test item showed dissection of the corneal stroma (eyeballs no. 1). The corneas had a normal histological structure in eyeball no 2 and no. 3.

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made.

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

The test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made.

Executive summary:

The isolated eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48 (GLP study). In the isolated chicken eye test (in vitro), toxic effects to the cornea were measured by a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention), a qualitative assessment of opacity, a quantitative measurement of increased thickness (swelling), and qualitative macroscopic and histopathological evaluations of morphological damage to the surface. The test item and the items used in the positive (10% acetic acid) and negative (physiological salt) controls in a volume of 0.03 mL were uniformly applied to the corneal surface. Three eyeballs were used for the test item and three for each control item. Every time, the test item and the control items were applied to the corneal surface for 10 seconds and kept at temperature between 20 – 23º C. Then, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature. The corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once (30 minutes after the end of the exposure). Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4% solution of formaldehyde in order to allow histopathological examinations to be conducted. The mean fluorescein retention score for the eyeballs treated with the test item was equal to 1.0 (ICE class II); the mean corneal opacity score for the eyeballs treated with the test item was equal to 1.0 (ICE class II); the mean corneal swelling value for the eyeballs treated with the test item were from 2.0 (I ICE class) to 6.9 (II ICE class).These results can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the eyeballs treated with the test item did not reveal any changes of the corneal surface. Histopathological examinations of the corneas treated with the test item showed dissection of the corneal stroma (eyeballs no. 1). The corneas had a normal histological structure in eyeball no 2 and no. 3. On the grounds of the study results and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 3xII.