4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

Endpoint summary

Description of key information

The test item showed no skin irritation potential in vitro.

The test material did not show any skin irritation potential in an in vivo study.

The test item did not show an ocular corrosive or severe irritant potential in vitro.

The test item caused no eye irritation or damage to the eye in an in vivo study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-01-11 to 2011-03-18

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)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

The test system used is a established standard model for in vitro skin irritation testing.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was not dissolved in a vehicle. However, the tissue surface was wetted with 10 µL deionised water before application.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The applied human in vitro skin model RHE was produced by SkinEthic Laboratories (Lyon, France).
- Tissue batch number: 11022A0209
No further details were specified.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1, using at least 25 mL PBS
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/L
- Incubation time: 3 h +/- 5 min
- Spectrophotometer: plate spectrophotometer, not further specified
- Wavelength: 570 nm
No further details were specified.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No details were specified.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The pretest for MTT-reducing capacity of the test item showed that the test item did not reduce MTT. Therefore, no controls were used.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is equal or less than 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is more than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
- Concentration: 5 % in deionised water

Duration of treatment / exposure:

42 +/- 1 min

Duration of post-treatment incubation (if applicable):

42 +/- 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value

Value:

126.44

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: MTT was not reduced by the test item.
- Colour interference with MTT: No change in color observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The optical density of negative control (mean value: 1.822 +/- 0.06 %) was >= 1.2 and standard deviation was <= 18 %.
- Acceptance criteria met for positive control: Yes. The relative cell viability of the positive control (mean value: 1.51, standard deviation 0.21 %) was < 40 % and standard deviation was <= 18 %.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation of 6.88 was <= 18 %.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.822	100
Positive Control	42	0.028	1.51
Test Material	42	2.303	126.44

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no skin irritation potential.

Executive summary:

The objective of the present study was to investigate potential of the test material to induce skin irritation in an in vitro human skin model. The test conducted according to OECD 439 consisted of a topical exposure of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (PBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before adding the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to the tissues. After treatment with the negative control the mean OD was 1.822 and thus >1.2. Treatment with the positive control revealed a mean viability value of 1.51 % (study acceptance criteria: < 40 %). Therefore, the study fulfilled the validity criteria. The mean tissue viability after treatment with the test material was 126.44 %, clearly exceeding 50 %. Therefore, the test material is considered as non-irritant to the skin in this in vitro assay.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-02-14 to 2013-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002-04-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 24 - 25 weeks
- Weight at study initiation: 4.10 kg
- Housing: separately
- Diet: ad libitum, Provimi Kliba 3418.0, ssniff meadow hay and additionally ssniff K snack
- Water: ad libitum, tap water
- Acclimation period:at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19
- Humidity (%): 50 - 59
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Remarks:

Aqua ad iniectabilia

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g
- Concentration: not dissolved but mixed with some drops of Aqua ad iniectabilia and grounded in mortar using a pestle.

Duration of treatment / exposure:

4 hours

Observation period:

8 days

Number of animals:

3 animals (females)

Details on study design:

TEST SITE
- Area of exposure: 6 cm^2
- % coverage: 100 %
- Type of wrap: Test item was applied to a gauze patch (Verbandmull ZZ from Paul Hartmann AG, Heidenheim, Germany) and fixed on the animals skin using non-irritating tape (Fixomull from BSN medical GmbH, Hamburg, Germany).

REMOVAL OF TEST SUBSTANCE
- Washing: no, but test item was wiped of using a dry cloth
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
after removal of the patches: 1, 24, 48, 72 hours, and then daily up to experimental day 8

SCORING SYSTEM:
- Method of calculation: Draize Scoring according to OECD and EC guidelines

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable as no effect observed

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable as no effect observed

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No signs of skin irritation were detected.

Other effects:

No mortality was observed.
Clinical examination revealed no clinical symptoms.
The body weight development during the study was inconspicuous in all treated rabbits.

Individual local findings

Animal	Findings	Readings after
		1 hour	24 hours	48 hours	72 hours	5 days	6 days	7 days	8 days
1	Erythema	0	0	0	0	0	0	0	0
	Edema	0	0	0	0	0	0	0	0
1	Erythema	0	0	0	0	0	0	0	0
	Edema	0	0	0	0	0	0	0	0
3	Erythema	0	0	0	0	0	0	0	0
	Edema	0	0	0	0	0	0	0	0

Skin irritation mean scores

Animal	Mean Scores		Max. Scores
	Erythema	Edema	Erythema	Edema
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test material did not show any skin irritation potential under the conditions of the present in vivo study.

Executive summary:

The study was performed to investigate the skin irritation potential of the test material in vivo. It was performed according to GLP and the methods applied are fully compliant with OECD 404. The test material was mixed with some drops of Aqua ad iniectabilia to ensure good contact with the skin. Afterwards, the test material was spread onto patches and applied to the intact skin of previously shaven rabbits for 4 hours under semi-occlusive conditions. The study was performed initially with one animal, followed by the confirmatory test with two further animals. The first examination of the treated skin followed 1 hour after patch removal. Thereafter, examinations were performed daily for further 7 days. No mortality occurred during the experimental phase of the study. The body weight development of the treated rabbits was inconspicuous. Clinical examination revealed no clinical symptoms and no signs of skin irritation. The skin irritation mean score 24, 48, and 72 hours after treatment and the maximum values were 0 for erythema and 0 for edema in all animals at all observations. The test material did not show any skin irritation potential under the conditions of the present in vivo study.

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:

weight of evidence

Study period:

2013-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009-09-01

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: from slaughterhouse (Odenwaldschlachthof Brensbach, Brensbach, Germany)
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium (HBSS, Hanks’ Balanced Salt Solution) with added Streptomycin and Penicillin (5 mL/ 500 mL HBSS). No further details provided.
- Time interval prior to initiating testing: Eyes were freshly isolated from donor cattle and were transported and immediately after delivery corneas were prepared.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were carefully examined macroscopically for defects (like vascularization, pigmentation, opacity or scratches). Those with defects were discarded. Furthermore, corneas were macroscopically checked after preparation and tissues with damages (e.g. scratches, pigmentation, neovarcularization) or opacity > 7 opacity units were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin were added in transport medium.
- Selection and preparation of corneas: The corneas were carefully removed from the eyes but a rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in incubation medium (EMEM, pre-warmed at 32 °C). Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O -ring) of the posterior part of the holder. The anterior part of the holder was positioned on top of the cornea. Both compartments of the holder were filled with incubation medium (EMEM). The posterior compartment was filled first to return the cornea to its natural convex form. For equilibration, the corneas in the holder were incubated (incubator: Grumbach BSS 160) in a vertical position at 32 ± 1 °C for about one hour. At the end of the incubation period, the incubation medium (EMEM) was removed from both compartments and replaced by fresh incubation medium (EMEM).
- Quality check of the isolated corneas: The baseline opacity was determined with an opacitometer (BASF-OP2.0). The opacitometer measured the light transmission passing through the corneas and displayed a lux value. This value is converted into an opacity value. Three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control groups.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20 %

VEHICLE
- Amount applied: 750 µL
- Concentration: 0.9 %
- Lot No: 12084014

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20 %

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

Incubation with fluorescein solution: 90 min

Number of animals or in vitro replicates:

3 replicates in all groups

Details on study design:

TREATMENT METHOD: closed chamber / open chamber, not specified

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times (or until no visual evidence of the test substance was observed)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity was determined with an opacitometer (BASF-OP2.0). The change in opacity was calculated by subtracting the initial baseline opacity from the post treatment opacity.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (BioTek ELx800) at 490 nm (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: according to TG

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-0.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative (vehicle) control: Yes, the calculated IVIS was 1.2 and, thus, within two standard deviations of the historical mean (IVIS: -0.6 to 3.9).
- Acceptance criteria met for positive control: Yes, the calculated IVIS was 81.6 and, thus, within two standard deviations of the historical mean (IVIS:76.6 to 142.7).

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative Control	0.9 % NaCl Solution	0.258	0.002	0.293	1.2	1.5
		2.907	-0.001	2.897
		0.364	0.000	0.369
Positive Control	20 % Imidazole solution	52.926	1.679	78.116	81.6	12.3
		49.082	1.494	74.492
		63.143	2.144	95.298
Test material	Test item	-1.962	0.000	-1.957	-0.8	1.0
		-0.182	-0.001	-0.192
		-0.178	-0.002	-0.208

Interpretation of results:

other: not corrosive, not severe irritant

Conclusions:

Under the conditions of the present study, the test item did not show an ocular corrosive or severe irritant potential in this in vitro assay.

Executive summary:

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment and was conducted according to OECD 437. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. As negative control 0.9 % sodium chloride solution and as positive control 20 % (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control the calculated IVIS was 1.2 and, thus, within two standard deviations of the historical mean (IVIS: -0.6 to 3.9). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 81.6 and was thus, within two standard deviations of the historical mean (IVIS:76.6 to 142.7). Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with test item was -0.8 and, thus, lower than 55. Under the conditions of the present study, the test item did not show an ocular corrosive or severe irritant potential in this in vitro assay.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-04-09 to 2013-04-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012-10-02

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL (NZW)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 32 - 33 weeks
- Weight at study initiation: 4.55 kg
- Housing: separately
- Diet: ad libitum, Provimi Kliba 3418.0, ssniff meadow hay and additionally ssniff K snack
- Water: ad libitum, tap water
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 52 - 63
- Air changes (per hr): not specified, but air conditioned room
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL
Before application the test item was ground in a mortar using a pestle.

Duration of treatment / exposure:

single treatment, eyes closed for 1 second

Observation period (in vivo):

The rabbits were examined for eye alterations, behavior, and general condition 1 hour and 6 hours after treatment and then twice daily up to experimental day 4.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: not done

SCORING SYSTEM: according to TG

TOOL USED TO ASSESS SCORE: not specified. 24 h before treatment animals were checked for normal eyes using hand-slit lamp and fluorescein. Only healthy animals were selected.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable as no effect observed

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable as no effect observed

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable as no effect observed

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable as no effect observed

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No signs of irritation were observed at the cornea or iris. Two animals showed redness of the conjunctivae (score 1) 1 h after instillation. The redness was completely reversible within 6 h after application.
No abnormalities were detected in the untreated eyes.

Other effects:

- Lesions and clinical observations: All animals survived the observation period. No clinical signs were seen during the experimental phase. Body weight development was inconspicuous in all treated animals.
No further effects were described.

Individual Findings (Scores) after test material application to the left eye:

Animal	Finding	Day
		1 (1h)	1 (6h)	2 (24 h)	2 (30 h)	3 (48 h)	3 (54 h)	4 (72 h)	4 (78 h)
1	Cornea (Opacity)	0	0	0	0	0	0	0	0
	Cornea (Area)	0	0	0	0	0	0	0	0
	Iris	0	0	0	0	0	0	0	0
	Conjuntivae	0	0	0	0	0	0	0	0
	Chemosis	0	0	0	0	0	0	0	0
	Discharge	0	0	0	0	0	0	0	0
2	Cornea (Opacity)	0	0	0	0	0	0	0	0
	Cornea (Area)	0	0	0	0	0	0	0	0
	Iris	0	0	0	0	0	0	0	0
	Conjuntivae	1	0	0	0	0	0	0	0
	Chemosis	0	0	0	0	0	0	0	0
	Discharge	0	0	0	0	0	0	0	0
3	Cornea (Opacity)	0	0	0	0	0	0	0	0
	Cornea (Area)	0	0	0	0	0	0	0	0
	Iris	0	0	0	0	0	0	0	0
	Conjuntivae	1	0	0	0	0	0	0	0
	Chemosis	0	0	0	0	0	0	0	0
	Discharge	0	0	0	0	0	0	0	0

Eye irritation Mean Scores and Max Score 24, 48 and 72 hours after application of the test material:

Animal	Mean Scores				Max Score
	Cornea (Opacity)	Iris	Conjuntivae	Chemosis	Cornea (Opacity)	Iris	Conjuntivae	Chemosis
1	0.00	0.00	0.00	0.00	0	0	0	0
2	0.00	0.00	0.00	0.00	0	0	0	0
3	0.00	0.00	0.00	0.00	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

For the test material, no eye irritating potential was detected under the conditions of the present in vivo study.

Executive summary:

A study according to OECD 405 was conducted to investigate the eye irritation potential of the test material in vivo. For this purpose the test material was applied in a single dose to one of the eyes of rabbits, the untreated eye served as control. The study was performed initially with one animal, followed by the confirmatory test with two further animals. The degree of eye irritation was evaluated by scoring lesions of conjunctiva, cornea and iris at specific intervals. The first examinations of the eyes followed 1 and 6 hours after treatment. Subsequently, the eyes were investigated twice daily for a further 4 days. In addition, potential systemic effects and the body weight development was determined. No mortality occurred and no clinical symptoms were seen during the experimental phase of the study. The body weight development of the rabbits was inconspicuous. No signs of irritation were observed at the cornea or iris. Two animals showed redness of the conjunctivae (score 1) 1 hour after instillation of the test material. The redness was completely reversible within 6 h after application. No abnormalities were detected in the untreated eyes. Eye irritation mean and maximum scores at 24, 48 and 72 hours after application of the test material were 0 for cornea opacity, iris, conjuntivae and chemosis. For the test material, no eye irritating potential was detected under the conditions of the present in vivo study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A step wise approach was used to address the skin irritation potential of the test material and is presented here as a weight of evidence approach. An in vitro study was conducted that resulted in no classification. Therefore, as required by a non-EU regulatory authority, an in vivo study was conducted. 

 

OECD 439

The objective of the present study was to investigate potential of the test material to induce skin irritation in an in vitro human skin model. The test conducted according to OECD 439 consisted of a topical exposure of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (PBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before adding the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to the tissues. After treatment with the negative control the mean OD was 1.822 and thus >1.2. Treatment with the positive control revealed a mean viability value of 1.51 % (study acceptance criteria: < 40 %). Therefore, the study fulfilled the validity criteria. The mean tissue viability after treatment with the test material was 126.44 %, clearly exceeding 50 %. Therefore, the test material is considered as non-irritant to the skin in this in vitro assay.

 

OECD 404

The study was performed to investigate the skin irritation potential of the test material in vivo. It was performed according to GLP and the methods applied are fully compliant with OECD 404. The test material was mixed with some drops of Aqua ad iniectabilia to ensure good contact with the skin. Afterwards, the test material was spread onto patches and applied to the intact skin of previously shaven rabbits for 4 hours under semi-occlusive conditions. The study was performed initially with one animal, followed by the confirmatory test with two further animals. The first examination of the treated skin followed 1 hour after patch removal. Thereafter, examinations were performed daily for further 7 days. No mortality occurred during the experimental phase of the study. The body weight development of the treated rabbits was inconspicuous. Clinical examination revealed no clinical symptoms and no signs of skin irritation. The skin irritation mean score 24, 48, and 72 hours after treatment and the maximum values were 0 for erythema and 0 for edema in all animals at all observations. The test material did not show any skin irritation potential under the conditions of the present in vivo study.

 

Conclusion

Both the in vitro study and the in vivo study showed no skin irritation potential of the test item. Therefore, the test item is considered to not cause skin irritation or corrosion. 

 

Eye irritation

A step wise approach was used to address the potential of the test material to cause damage or irritation to the eye and is presented here as a weight of evidence approach. An in vitro study was conducted that resulted in no classification. Therefore, as required by a non-EU regulatory authority, an in vivo study was conducted. 

 

OECD 437

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment and was conducted according to OECD 437. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. As negative control 0.9 % sodium chloride solution and as positive control 20 % (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control the calculated IVIS was 1.2 and, thus, within two standard deviations of the historical mean (IVIS: -0.6 to 3.9). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 81.6 and was thus, within two standard deviations of the historical mean (IVIS:76.6 to 142.7). Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with test item was -0.8 and, thus, lower than 55. Under the conditions of the present study, the test item did not show an ocular corrosive or severe irritant potential in this in vitro assay.

 

OECD 405

A study according to OECD 405 was conducted to investigate the eye irritation potential of the test material in vivo. For this purpose the test material was applied in a single dose to one of the eyes of rabbits, the untreated eye served as control. The study was performed initially with one animal, followed by the confirmatory test with two further animals. The degree of eye irritation was evaluated by scoring lesions of conjunctiva, cornea and iris at specific intervals. The first examinations of the eyes followed 1 and 6 hours after treatment. Subsequently, the eyes were investigated twice daily for a further 4 days. In addition, potential systemic effects and the body weight development was determined. No mortality occurred and no clinical symptoms were seen during the experimental phase of the study. The body weight development of the rabbits was inconspicuous. No signs of irritation were observed at the cornea or iris. Two animals showed redness of the conjunctivae (score 1) 1 hour after instillation of the test material. The redness was completely reversible within 6 h after application. No abnormalities were detected in the untreated eyes. Eye irritation mean and maximum scores at 24, 48 and 72 hours after application of the test material were 0 for cornea opacity, iris, conjuntivae and chemosis. For the test material, no eye irritating potential was detected under the conditions of the present in vivo study.

 

Conclusion

Both the in vitro study and the in vivo study showed no eye irritation potential of the test item. Therefore, the test item is considered to not cause damage or irritation to the eye. 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on available data on skin irritation/corrosion, the test item does not require classification for causing skin irritation or corrosion according to Regulation (EC) No 1272/2008 (CLP).

Based on available data on eye irritation/corrosion, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP).