Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Endpoint summary

Description of key information

Skin irritation: No adverse skin irritation effects were observed in all tests.

Eye irritation: No acute eye irritation potential was determined in a study according to OECD guideline No. 437

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:

key study

Study period:

15 February 2016 - 22 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

According to Annex VII of the REACH Regulation, if new test data are required these must be derived from in vitro methods only.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Based on a "Statement on the Scientific Validity of In Vitro Tests for Skin Irritation" (November 2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

3rd revision (2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Source strain:

other:

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Human donation
- Tissue: normal epidermal keratinocytes

Justification for test system used:

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. Skin irritation is defined in Section 3.2.1.1 of Annex I to the CLP regulation as “...the production of reversible damage of the skin following the application of a test substance for up to 4 hours”. The EpiDerm™ human skin model (OECD 439) is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT Kit
- Tissue batch number(s): 23314
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 16 February 2016
- Date of initiation of testing: Pre-incubation phase started 16 February 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 ºC
- Temperature of post-treatment incubation (if applicable): 37 ± 1 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 300 μL, one washing step
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Not applicable, microplate reader used
- Wavelength: 570 ± 1 nm
- Filter: Not reported
- Filter bandwidth: 570 ± 1 nm
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control; 4.77 %
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: None reported
- Reproducibility: Relative standard deviation for mean viability; 14.79 % postive control. Relative standard deviation for mean absorption; 16.6 % postive control, 8.55 % negative control.

NUMBER OF REPLICATE TISSUES: 3 per control and test item treatment

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 43.5 hours exposure is less than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): Undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5 % SLS in deioinsied water; concentration not reported

Duration of treatment / exposure:

43.5 hours

Duration of post-treatment incubation (if applicable):

67.5 hours

Number of replicates:

3 per test item, negative or positive control

Irritation / corrosion parameter:

other: mean tissue absorbance

Remarks:

Of three wells after blank corrction

Run / experiment:

1

Value:

1.615

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Mean tissue absorbance

Remarks:

Of three wells after blank

Run / experiment:

2

Value:

1.338

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Mean tissue absorbance

Remarks:

Of three wells after blank correction

Run / experiment:

3

Value:

1.495

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Mean tissue absorbance

Remarks:

Mean of three tissue after blank correction

Run / experiment:

Mean

Value:

1.483

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (%)

Remarks:

compared to blank

Run / experiment:

TIssue

Value:

103.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (%)

Remarks:

compared to blank

Run / experiment:

Tissue 2

Value:

85.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (%)

Remarks:

compared to blank

Run / experiment:

Tissue 3

Value:

95.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Mean relative abosrbance

Remarks:

% of negative control

Run / experiment:

Mean

Value:

94.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Did not show blue colour after 1 hour incubation
- Colour interference with MTT: No colour change

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, within the required range of OD ≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: Yes, induced a decrease in absorbance of 5.1 % compared to the negative control
- Acceptance criteria met for variability between replicate measurements: Not reported
- Range of historical values if different from the ones specified in the test guideline:Not applicable

Results of treatment with test item and the controls

Dose Group	Exposure Interval	Tissue No.	Absorbance 570 nm (Well 1)	Absorbance 570 nm (Well 2)	Absorbance 570 nm (Well 3)	Mean Absorbance of 3 Wells*	Mean Absorbance of three wells blank corrected	Mean Absorbance of three tissues after blank correction	Rel. Absorbance (%) 1, 2 + 3**	Relative Standard Deviation (%)	Mean. Rel. Absorbance (% of negative control)***
Blank			0.037	0.038	0.038	0.038	0.000
Negative Control	60 mins	1	1.540	1.548	1.508	1.532	1.495	1.563	95.6	4.1	100.0
		2	1.644	1.591	1.593	1.609	1.572		100.6
		3	1.717	1.631	1.630	1.659	1.622		103.8
Positive Control	60 mins	1	0.117	0.120	0.118	0.118	0.081	0.080	5.2	4.4	5.1
		2	0.118	0.107	0.116	0.113	0.076		4.8
		3	0.104	0.132	0.124	0.120	0.083		5.3
Test item	60 mins	1	1.672	1.634	1.652	1.653	1.615	1.483	103.4	9.4	94.9
		2	1.384	1.362	1.380	1.375	1.338		85.6
		3	1.530	1.528	1.540	1.532	1.495		95.7

 * Mean of three replicate wells after blank correction

** relative absorbance per tissue (rounded values): 100*(absorbance_tissue)/ (mean absorbance_{negative control})

***relative absorbance per treatment group (rounded values): 100*(mean absorbance_{test item/positive control})/ (mean absorbance_{negative control})

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was non-cytotoxic in an experiment employing an artificial three-dimensional model of human skin. Conducted according to OECD 439 (EU B.46) and GLP, the in vitro study is considered reliable without restriction (Klimisch 1). The test item should not be classified as an irritant (UN GHS no category).

Executive summary:

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (94.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.   The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 March 2013 - 15 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Non-GLP study conducted in accordance with Declaration of Helsinki, the harmonised ICH Guideline E6 for Good glinical practice and the requirements of 21 CFR Parts 50 and 56. Consumer Product Testing Company (CPTC) is audited by a Quality Assurance Unit. Full study records are stored for a minimum of 10 years.

Justification for type of information:

Whilst Annex VII does not require in vivo testing for skin corrosion/irritation, all existing available information should be evaluated, including any available human data. The Human Patch Test is a controlled study involving the exposure of small patches of skin to the test item. Whilst the CLP regulation does not contain clear criteria for classification for skin irritation based on human data, the studies provide useful information on skin irritancy.

Qualifier:

according to guideline

Guideline:

other: ICH Guideline E6

Principles of method if other than guideline:

Study was conducted according to CPTC procedure CP-01.02

GLP compliance:

no

Remarks:

Good Clinical Practice (GCP)

Species:

human

Details on test animals or test system and environmental conditions:

PARTICIPANTS: 52 male and female subjects >16 years of age

INCLUSION CRITERIA:
- male and female subjects aged 16 and over
- absence of visible skin disease
- prohibition of use of topical systemic steroids and/or antihistamines for 7 days prior to study initiation (acclimitisation)
- complete medical history and informed consent
- considered reliable and capable of following directions

EXCLUSION CRITERIA:
- ill health
- recieving medical attention which could influence the outcome of the study
- females who are pregnant or nursing
- a history of adverse reactions to topical ointments

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

other: control patch

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.2ml of 4% solution

Duration of treatment / exposure:

The test material remained in contact with the skin for a total of 48-hours.

Observation period:

Observations by dermatologist at 48 and 72 hours

Number of animals:

53 subjects (one did not complete the test for reasons unrelated to the use of the test material)

Details on study design:

Human skin patch testing aims to determine the primary irritation potential via epidermal contact. Small quantities (0.2ml) of test material were applied to occlusive patches of 3/4" x 3/4" absorbant pads in adhesive dressing between the scapulae on the upper back and sealed with a hypoallergenic adhesive tape. The test material remained in contact with the skin for a total of 48 hours. Signs of irritation were recorded by a dermatologist at 48 and 72 hours. The evaluation criteria included edema, dryness, staining, papules, vesicles, bullae, ulceration and spreading. The erythema was scored and recorded according to: no visible skin reaction (0); barely perceptible (0.5); mild (1); moderate (2); marked (3); or severe (4).

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Observations remained negative throughout the test interval. There were no visible skin reactions or adverse events.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

Conducted according to ICH Guideline E6 for Good Clinical Practice, the HPT was considered reliable without restriction. Whilst the CLP regulation does not contain clear criteria for classification for skin irritation based on human data, no visible skin reactions or adverse events were recorded. Consequently, there is not evidence of intrinsic corrosive or irritant properties requiring classification or substance specific risk mitigation measures (RMMs).

Executive summary:

The Human Patch Test is a controlled study involving the exposure of small patches of skin to the test item.Small quantities (0.2ml) of test material were applied to occlusive patches between the scapulae on the upper back. Signs of irritation, such as edema, dryness, staining, papules, vesicles, bullae, ulceration and spreading were recorded at 48 and 72 hours. Erythema was scored and recorded according to: no visible skin reaction (0); barely perceptible (0.5); mild (1); moderate (2); marked (3); or severe (4). All participants that completed the study showed no visible skin reaction (52/53 subjects). Under the conditions of the study, the test material did not indicate a potential for dermal irritation.

Reference 3

Endpoint:

skin irritation / corrosion

Remarks:

in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 May 1994 - 08 June 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

OECD TG 404 provides information on the health hazards likely to arise following dermal application. Originally adopted in 1981, the test guideline was revised in 1992, 2002 and 2015 to include guidance on integrated approaches to testing and assessment. Contrary to the current guideline, the study applied a diluent of the test item (30% in DPG) to the shaved dorsal test site.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Adopted July 17, 1992

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The test item was tested at 30% in DPG

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: Young albino rabbits
- Source: Commerical supplier
- Housing: Housed individually without bedding in cages (area 2450 cm2, height 42cm2)
- Acclimatisation: Animals were acclimated to the test conditions for 5 days prior to administration
- Preparation: A 3 x 5 cm2 area of the dorsal trunk area was shaved on the fifth day.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): 30
- Photoperiod (hrs dark / hrs light): 12:12

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

other: Dipropylene glycol (DPG)

Controls:

other: Control skin patch area

Amount / concentration applied:

The test item (30% in DPG) was dermally applied in a single dose of 0.5 ml/animal onto the shaved dorso-lumbar region of three albino rabbits. The treated region was protected with the non-stripped, non-irritating plastic material of the reverse of a patch.

Duration of treatment / exposure:

The treatment was held in place for 4 hours by an occlusive bandage made from an adhesive dressing. The occlusive bandage was removed after 4 hours and the residual test substance was removed via gently washing with water.

Observation period:

Animals were observed from the 25th May to the 8th June (1994) for toxicological symptoms, behaviour, body weight, skin reactions and mortality.

Number of animals:

3

Details on study design:

Evaluations of skin reactions were scored according to: no erythema (0); very slight erythema (barely perceptible) (1); well-defined erythema (2); moderate to severe erythema (3); and severe erythema to eschar formation preventing grading of erythema (4).

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0 - 0

Max. score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Dermal application of 0.5 ml test item diluent (30% in DPG) per animal did not induce corrosion, irritation or mortality in any of the three rabbits tested. Throughout the 14-day observation period, none of the animals showed signs of toxicity, altered behaviour or body weight. Two rabbits transiently showed very slight erythema (score 1) for 1-3 days following treatment. These erythemata could not be observed any more on day 5. No rabbit showed an oedema during the observation period. Desquamation of the skin of two rabbits could be observed from day 5 to the end of the observation period. Desquamation of the epidermis is common following injury of the skin.

Table 1. Acute dermal irritation/corrosion - Mean value of all animal skin reactions

Time after application	Test Substance		Control
	Erythema	Oedema	Erythema	Oedema
1 hour	0	0	0	0
Day 1	0.7	0	0	0
Day 2	0.7	0	0	0
Day 3	0.7	0	0	0
Day 4	-	-	-	-
Day 5	0	0	0	0
Day 6	0	0	0	0
Day 7	0	0	0	0
Day 8	0	0	0	0
Day 9	0	0	0	0
Day 10	-	-	-	-
Day 11	-	-	-	-
Day 12	0	0	0	0
Day 13	0	0	0	0
Day 14	0	0	0	0

 

Interpretation of results:

GHS criteria not met

Conclusions:

OECD TG 404 provides information on the health hazards likely to arise following dermal application. Conducted according to OECD TG 404 and GLP, the study was considered reliable with restriction (Klimisch 2). No signs of corrosion or irritancy were reported and there is no evidence of intrinsic properties requiring classification.

Executive summary:

Conducted according to OECD TG 404 Acute Dermal Toxicity, the GLP compliant study is considered reliable with restriction (Klimisch 2). Four-hour dermal exposure to a diluent of the test item (0.5 mL of 30% in DPG) did not induce corrosion, irritation or mortality in any of the three rabbits tested. Throughout the 14-day observation period, none of the animals showed signs of toxicity or altered behaviour. Two rabbits transiently showed very slight erythema (score 1) for 1-3 days following treatment, however, erythemata could not be observed on day 5. No rabbit showed an oedema during the observation period. Consequently, there is no evidence of intrinsic corrosive or irritant properties requiring classification or substance specific risk mitigation measures (RMMs).

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

July 2013

Deviations:

yes

Remarks:

The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details

Version / remarks:

April 1997

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine eyes

Strain:

other: Cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH Aschaffenburg
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): At least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):Isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled abattoir until transportation on the same morning to the laboratory.
- Time interval prior to initiating testing: The corneae were isolated on the same day as delivery
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects with those presenting defects discarded.
- Indication of any antibiotics used: Eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): Neat test item

VEHICLE: not applicable

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were carefully examined macroscopically for defects with those presenting defects discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue was left for stability and handling of the isolated cornea. Each isolated cornea was mounted in a specially designed cornea holder according to the OECD 437 guideline 437. The endothelial side of the cornea was positioned against the sealing ring of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium with the posterior compartment filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS: At the end of the incubation period, the basal opacity was determined (t0). Cornea with a score of > 7 was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Saline (0.9% NaCl in deionised water, using ultrasonic technique)

POSITIVE CONTROL USED: 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of neat test item

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:1
- POST-EXPOSURE INCUBATION:2 hours at 32 ± 1 ºC

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry(OD490)
- Others (e.g, pertinent visual observations, histopathology): None reported

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: According to guideline

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 1

Value:

0.05

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 2

Value:

-0.22

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 3

Value:

1.27

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.37

Vehicle controls validity:

not applicable

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: None reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Validity criteria met

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Not applicable

Results after 10 minute treatment time:

Test Group	Opacity value = Difference (t130 - t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy score
		Mean		Mean
Negative control	0	0.00	0.085	0.072	1.28	1.09	Not categorised
	0		0.067		1.01
	0		0.065		0.98
Positive control	96.00*		1.152*		113.28	103.28	Category 1
	83.00*		1.501*		105.51
	77.00*		0.938*		91.07
Test item	0.00*		0.004*		0.05	0.37	Not categorised
	0.00*		-0.014*		-0.22
	1.00*		0.018*		1.27

 *corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.37.
According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (GHS).

Executive summary:

The test item was assessed for eye irritation in a Bovine Corneal Opacity and Permeability Assay (BCOP) conducted following OECD

437, and EU B.47 in undiluted form alongside a postive and negative control. The results of the test indicates that the test item does not possess an eye irritating potential according to the UN GHS regulations.

The study is a GLP compliant guideline experiment study available as an unpublished study report. The study has no restrictions and is fully adequate for assessment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Key

In-vitro skin irritation test

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. The EpiDerm™ human skin model (OECD 439) is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP). Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (94.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin. The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).

Supporting

In support of the REACH Annex VII information requirement for skin corrosion/irritancy, two in vivo studies assessed the corrosion/irritancy of 4% and 30% dilutions of the test item, in a Human Patch Test and OECD TG 404 rabbit study, respectively. No signs of corrosion or irritancy were reported. Consequently, there is no evidence of intrinsic corrosive or irritant properties requiring classification or substance specific risk mitigation measures (RMMs).

Eye irritation

Key

The test item was assessed for eye irritation in a Bovine Corneal Opacity and Permeability Assay (BCOP) conducted following OECD 437, and EU B.47 in undiluted form alongside a postive and negative control. The results of the test indicates that the test item does not possess an eye irritating potential according to the UN GHS regulations.

The study is a GLP compliant guideline experiment study available as an unpublished study report. The study has no restrictions and is fully adequate for assessment.

Justification for classification or non-classification

Skin Irritation

 

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. As a result the substance is not considered to be classified for skin irritation under Regulation (EC) No 1272/2008

Eye irritation

 

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. As a result the substance is not considered to be classified for eye irritation under Regulation (EC) No 1272/2008