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

Endpoint summary

Description of key information

Skin irritation/corrosion: Test method according to OECD Guideline 430. GLP study. The test substance does not lead to skin corrosion/severe irritation.
Eye irritation: Test method according to OECD Guideline 438. GLP study. The test item did not cause eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2014 - 28 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 430. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

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 (incl. QA statement)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre for Experimental Medicine at the Medical University (Katowice).
- Age at study initiation: 23 days old
- Housing: plastic cage covered with a wire bar lid. Dimensions: 58 x 37 x 21 cm.
- Diet (e.g. ad libitum): ad libitum, "Murigran" standard granulated laboratory fodder.
- Water (e.g. ad libitum): ad libitum, drinking tap water.
- Acclimation period: 3 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21°C
- Humidity (%): 44 – 63%
- Air changes (per hr): about 16 times/hour.
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark.

Type of coverage:

other: Not applicable: in-vitro test.

Preparation of test site:

other: Not applicable: in-vitro test.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): undiluted test item in a volume of 150 μL

Duration of treatment / exposure:

24 hours

Number of animals:

2

Irritant / corrosive response data:

On the grounds of the study, it may be stated that the test item belongs of a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Table 1. Results of the control transcutaneous electrical resistance test (TER):

 

Animal number	Skin disc number	TER value (kΩ)
1	1	15.38
	2	14.18
2	1	18.31
	2	14.25

The skin disc resistance values were greater than 10 kΩ; therefore, the remainder of the animals’ skin discs could have been used in the experiment.

 

Table 2. Results of the transcutaneous electrical resistance test (TER):

 

Animal number	Tested substance	Skin disc number	TER value (kΩ)	Mean TER value ± SD (kΩ)
1	Positive control – 36% HCl	1	0.88	0.87 ± 0.02
		2	0.85
		3	0.87
	Negative control – distilled water	1	14.37	14.45 ± 0.54
		2	13.95
		3	15.03
	Test item	1	19.08	19.61 ± 0.47
		2	19.76
		3	19.98
2	Positive control – 36% HCl	1	0.95	0.93 ± 0.02
		2	0.91
		3	0.93
	Negative control – distilled water	1	16.38	17.30 ± 0.96
		2	17.21
		3	18.30
	Test item	1	19.07	19.21 ± 0.61
		2	18.68
		3	19.88

 

The concurrent mean values for the positive and negative controls were within the acceptable ranges for the method:

Positive control: 0.5-1.0 kΩ

Negative control: 10 -25 kΩ

The mean TER results for the skin discs treated with the test item were equal to 19.61 kΩ (animal no. 1) and 19.21 kΩ (animal no. 2).

 

Table 3. Gross changes on the surface of the treated skin discs:

 

Animal number	Tested substance	Skin disc number	Gross changes
1	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes
2	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes

 

The gross examination showed that the positive control skin discs exhibited skin perforation, whereas the negative control skin discs and the ones treated with the test item did not reveal any changes.

 

Interpretation of results:

other: non-corrosive.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test substance does not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 and EU Method B.40. (GLP study). Skin discs used in the experiment were obtained from two 30-day-old animals. The undiluted test item was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Positive (36% hydrochloric acid) and negative (distilled water) controls were conducted concurrently. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water. Prior to measuring the electrical resistance, the surface tension of the skin was reduced by adding a sufficient volume of 70% ethanol. After removing the ethanol the tissue was hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. The skin discs were subjected to a gross examination in order to reveal possible damage. The mean TER results for the skin discs treated with the test item were equal to 19.61 kΩ (animal no. 1) and 19.21 kΩ (animal no. 2). They can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs treated with the test item did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item does not lead to skin corrosion/severe irritation as the mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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.

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion: Key study: The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 and EU Method B.40. (GLP study). Skin discs used in the experiment were obtained from two 30-day-old animals. The undiluted test item was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Positive (36% hydrochloric acid) and negative (distilled water) controls were conducted concurrently. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. The skin discs were subjected to a gross examination in order to reveal possible damage. The mean TER results for the skin discs treated with the test item were equal to 19.61 kΩ (animal no. 1) and 19.21 kΩ (animal no. 2). Gross examinations of the skin discs treated with the test item did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item does not lead to skin corrosion/severe irritation as the mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Eye irritation: Key study: The isolated eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48 (GLP study). 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, an histopathological examination was 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). 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.

Justification for selection of skin irritation / corrosion endpoint:
Only one study is available.

Justification for selection of eye irritation endpoint:
Only one study is available.

Justification for classification or non-classification

Based on the available information, the substance is not classified for irritation/corrosion according to CLP Regulation (EC) no. 1272/2008.