3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

Endpoint summary

Description of key information

Skin irritation, in vitro: potentially irritating (15 minutes exposure/42hour incubation viability = 50.5%), EPISKIN, OECD TG 439, 2010

Skin irritation, in vivo (rabbit): irritating, EU Method B.4, 2010

 

Weight of evidence: eye irritating

Eye irritation, in vitro: study result: negative – however cannot be used as standalone assay (10 minute: relative viability = 85% and >50% not all acceptability criteria met), SkinEthic RHE test, eq. or similar to OECD TG 492, 2010

Eye irritation, in vitro enucleated (rabbit) eye (REET): potentially eye irritating, pre-test screening to in vivo, EU Method B.5, 2010

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

other justification

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex VII, column 2 section 8.1.1 (as amended by Commission Regulation (EU) 2016/863) the in vitro skin corrosion (OECD TG 431) study does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. An available in vivo (OECD TG 404) skin irritation study is available and data in other endpoints (such as skin sensitisation and/or acute dermal toxicity) indicates that the substance is not skin corrosive and a definitive conclusion on the classification can be made. Furthermore, in accordance with section 1.2 of REACH Regulation (EC) No. 1907/2006 Annex XI the weight of evidence indicates that the substance is not skin corrosive and therefore in vitro testing may be omitted. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.2, July 2017) the study does not need to be conducted.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-03-2010 to 30-06-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2009 ; signature: November 2009

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised Supplier (documented in the full study report)
- Age at study initiation: 12 to 20 weeks
- Weight at study initiation: 2.19 to 2.22 kg
- Housing: Individually housed in suspended cages.
- Diet (ad libitum): Certified Rabbit diet (recognised supplier); provided ad libitum
- Water (ad libitum): mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 to 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 23-03-2010 To: 06-04-2010

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL
- Concentration (if solution): Not applicable.

VEHICLE
- Amount applied: Not applicable.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours (initial observation); additional observations are made daily up to Days 7 and 14 to assess the reversibility of skin reactions (as appropriate).

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: dorsal
- Type of wrap if used: semi-occlusive (2.5 cm x 2.5 cm cotton gauze patch secured with surgical adhesive tape)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours. Earlier if appropriate based on observations.

OBSERVATION TIME POINTS
(indicate if minutes, hours or days): 1 hour, 4 hours, 24 hours, 48 hours and 72 hours. Additional observations daily up to 7 or 14 days, as appropriate

SCORING SYSTEM: Draize Scale:
Erythema and Eschar Formation
No erythema _______________________________________________________ 0
Very slight erythema (barely perceptible) __________________________________ 1
Well-defined erythema ________________________________________________ 2
Moderate to severe erythema ___________________________________________3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) _______ 4

Oedema Formation
No oedema ________________________________________________________________ 0
Very slight oedema (barely perceptible) ___________________________________________ 1
Slight oedema (edges of area well-defined by definite raising) __________________________ 2
Moderate oedema (raised approximately 1 millimetre) _______________________________ 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) ___4
Any other skin reactions and clinical signs of toxicity, if present, were also recorded.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: Slight desquamation at day 14

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks:

Slight desquamation at day 14

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks:

Slight desquamation at day 14

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

Well-defined erythema (score =2) and slight oedema (score = 2) were noted at both (n=2) treated skin sites at the 24, 48 and 72-Hour observations. Other skin reactions noted at both treated skin sites at the 48 and 72-Hour observations were light brown discolouration of the epidermis and loss of skin flexibility and/or elasticity. Crust formation, prevented accurate evaluation of erythema and oedema, was noted at both treated skin sites at the 7-Day observation. Slight desquamation was noted at both treated skin sites at the 14-Day observation.

Other effects:

- Other adverse local effects: None reported.
- Other adverse systemic effects: None reported. Individual bodyweights increased during the conduct of the study period.

Table 1. Individual skin reactions

Skin Reaction	Observation Time	Individual Scores
		1 (male)	2 (male)	3 (-)
Erythema/Eschar formation	1 hour	0	0	-
	24 hours	2	2	-
	48 hours	2 BrLe	2 BrLe	-
	72 hours	2 BrLeLf	2 BrLeLf	-
	7 days	#e Cf	#e Cf	-
	14 days	0 D	0 D	-
Oedema formation	1 hour	0	0	-
	24 hours	2	2	-
	48 hours	2	2	-
	72 hours	2	2	-
	7 days	#od	#od	-
	14 days	0	0	-

Br = Light brown discoloration of the epidermis

Le = Loss of skin elasticity

Lf = Loss of skin flexibility

Cf = Crust formation

D = Slight desquamation

#e = adverse reaction prevented accurate evaluation of erythema

#od = adverse reaction prevented accurate evaluation of oedema

- = observation not performed or observation not required

 

Mean scores per organism at 24, 48 and 72h:

Erythemea/Escar Formation:

1: total = 6 ; mean score = 2.0 (and slight desquamation at day 14)

2: total = 6 ; mean score = 2.0 (and slight desquamation at day 14)

3: total = - ; mean score = - (score -)

Oedema Formation:

1: total = 0 ; mean score = 2.0 (reversed at day 14)

2. total = 5 ; mean score = 2.0 (reversed at day 14)

3: total = - ; mean score = - (score -)

 

Note: Crust formation, prevented accurate evaluation of erythema and oedema, was noted at both treated skin sites at the 7-Day observation. Slight desquamation was noted at both treated skin sites at the 14-Day observation.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is considered to be irritating.

Executive summary:

The study was performed to OECD TG 404 and to assess the primary skin irritancy potential of the test item in New Zealand White rabbits in accordance with GLP. The test item was applied to two males f by semi-occluded application to the intact rabbit skin with 0.5 mL test item introduced under a 2.5 cm x 2.5 cm cotton gauze patch on the clipped skin or 4-hours. The patch was secured in position with a strip of surgical adhesive tape. After exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, and 72 hours. No corrosive effects were noted. Well-defined erythema (score =2) and slight oedema (score = 2) were noted at both (n=2) treated skin sites at the 24, 48 and 72-Hour observations. Other skin reactions noted at both treated skin sites at the 48 and 72-Hour observations were light brown discolouration of the epidermis and loss of skin flexibility and/or elasticity. Crust formation, prevented accurate evaluation of erythema and oedema, was noted at both treated skin sites at the 7-Day observation. Mean scores following grading at 24, 48 and 72h were score 2.0/2.0 in erythema and eschar and score 2.0/2.0 in oedema scoring criteria, respectively. Slight desquamation was noted at both treated skin sites at the 14-Day observation. Under the conditions of the study, the test item is considered to be a skin irritant. Applicant assessment indicates: that the CLP Regulation (EC) 1272/2008 Skin Irritation : category 2 classification within the inflammation criteria (erythema/eschar and oedema) being mean score at 24, 48 and 72 h > or = 2.3 in two (of three potential) sites was not met. The study was originally classified under DSD 67/548/EEC which utilised a mean score of 2.0 for positive result. The study was terminated on day 14 under DSD 67/548/EEC, all erythema/eschar and oedema inflammation had ceased although ‘slight desquamation’ persisted in two (of three potential) sites to day 14. Therefore, CLP Regulation (EC) 1272/2008 : Skin Irritant Category 2 classification is maintained.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

key study

Study period:

02-03-2010 to 19-05-2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Conducted equivalent to or similar to guideline and performed under GLP. The results of the assay should not be utilised as a standalone assay.

Qualifier:

according to guideline

Guideline:

other: SkinEthic™ HCE EIT

Version / remarks:

Study conducted prior to the full validation and/or OECD acceptance of the test assay ; however conducted to equivalent methodology, scientifically valid with sufficient documentation suitable for assessment.
The development of the model is documented in : Nguyen D. et al., Three-dimensional construct of the human corneal epithelium for in vitro toxicology. In: H. Salem and S.A. Katz, Editors, Alternative Toxicological Methods, CRC Press (2003), pp.147-159.
The prevalidation was documented in: Van Goethem F., et al., Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals. Toxicology in vitro 20 (2006), pp.1-17.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

SkinEthic™ HCE EIT

Deviations:

yes

Remarks:

Sodium Dodecyl Sulphate (SDS) 1%w/v was used as the positive control rather than Methyl Acetate. The expsoure duration was 10 minutes rather than 30 minutes in the modern guideline. The results of the assay should not be utilised as a standalone assay.

Qualifier:

equivalent or similar to guideline

Guideline:

other: EURL ECVAM DB-ALM: Protocol no. 190 : SkinEthic HCE Eye Irritation Test Liquid (EITL)

Version / remarks:

SkinEthic™ HCE EIT

Deviations:

yes

Remarks:

Sodium Dodecyl Sulphate (SDS) 1%w/v was used as the positive control rather than Methyl Acetate. The expsoure duration was 10 minutes rather than 30 minutes in the modern guideline. The results of the assay should not be utilised as a standalone assay.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2009 ; signature: November 2009

Species:

other: human-derived epidermal keratinocyte tissues

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The test method, subsequent to the conduct of the test is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: SkinEthic RHC model (otherwise known as the SkinEthic HCE model) : consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The supplier of the test tissues is SkinEthic Laboratories, France. Date of receipt by the test laboratory was on the < 24 hours prior to the conduct of the test.

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): 30 µL of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

Duration of treatment / exposure:

10 mins at 37.0 ± 1.0°C, 5% CO2 in air

Duration of post- treatment incubation (in vitro):

3 hours at 37.0 ± 1.0°C, 5% CO2 in air post treatment / rinsing incubation
- Rinsing consisted of initially rinsing each tissue insert using DPBS. Rinsing was achieved by filling and emptying tissue Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed.
- Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µl of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air.

Number of animals or in vitro replicates:

Two tissues were used for each treatment group (test item, negative control and positive control).

Details on study design:

- Details of the test procedure used: See below.
- RhCE tissue construct used, including batch number: SkinEthic RHC model (otherwise known as the SkinEthic HCE model) date of receipt provided in the full study report.
- Doses of test chemical and control substances used: 30 µL of the test item / 30 µL “Solution A” used as supplied by manufacturer (negative control) / 30 µL Sodium Dodecyl Sulphate (SDS) 1%w/v in sterile distilled water (positive control)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Duplicate tissues were treated with 30 µL of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µl of solution A to serve as negative controls and triplicate tissues were treated with 30 µL of 1% w/v SDS to serve as positive controls. The plates were incubated at 37°C, 5% CO2 in air during the exposure time. Rinsing consisted of initially rinsing each tissue insert using DPBS. Rinsing was achieved by filling and emptying tissue Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air. At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminum foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): "Solution A" : used as supplied ; composition per litre : Na2HPO4 0.142 g/L; Glucose 1.802 g/L ; HEPES 7.149 g/L ; KCl 0.224 g/L ; NaCl 7.597 g/L ; Two tissues were used for the control group. This solution was provided in accordance with the test kit manufacturer instructions.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable. Sodium Dodecyl Sulphate (SDS) 1%w/v was used as the positive control rather than Methyl Acetate ; this was a suitable PC according to the test method at the time. Does not impact the interpretation of the study.
- Description of any modifications to the test procedure: Not applicable.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
Colour-interference: was not checked. However the test item is colourless and it can be considered that colour-interference is not applicable.
Direct reduction of MTT: was checked. The test item was checked for possible direct MTT reduction before the definitive test. 30 µL of test item was added to a 0.5 mg/mL MTT solution and incubated at room temperature in the dark for 60 minutes. Untreated MTT solution was used as a control. If the MTT solution turned black/purple, the test material was presumed to have reduced the MTT. If the MTT solution colour turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay. Conclusion: the test item did not directly interact with MTT and therefore considered was not a potential direct MTT reducer. As a result: NSMTT, NSCliving and NSCkilled were not required.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Duplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: Athos 2001 microplate reader – spectrophotometrically (at 540 nm).
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: 60% viability cut off, according to the manufacturer instructions. However, later applications of the method indicate to utilise the 50% cut off per the OECD TG 492 and GHS system.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC rather than later proscribed Ca2+/Mg2+-free DPBS. The NC was tested
concurrently. The difference in viability was < 10% between replicates. The Positive Control gave a response within an expected PC range mean = 39% (positive) tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Therefore the results of the assay should be interpreted with additional caution and not as a standalone assay.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data was not available in the study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is (post study) validated. The test laboratory had previously demonstrated technical proficiency and this information is in the public domain.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: < 10 % in NC. >20% in PC. The results of the assay should however be interpreted with additional caution and not as a standalone assay.
- Acceptable variability between tissue replicates for the test chemical: Yes.

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean (n=2) - test item

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC

Positive controls validity:

other:

Remarks:

Positive Control gave a response within an expected PC range mean = 39% (positive) tissue range : 24 – 53%. The difference in viability was > 20% between replicates. See Remarks.

Remarks on result:

other: - Acceptable variability between tissue replicates for positive and negative controls: < 10 % in NC. >20% in PC. The results of the assay should however be interpreted with additional caution and not as a standalone assay.

Remarks:

See positive control results

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean (n=2) - positive control

Value:

39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC

Positive controls validity:

other: Positive Control gave a response within an expected PC range mean = 39% (positive) tissue range : 24 – 53%. The difference in viability was > 20% between replicates. See Remarks.

Remarks on result:

positive indication of irritation

Remarks:

- Acceptable variability between tissue replicates for positive and negative controls: < 10 % in NC. >20% in PC. The results of the assay should however be interpreted with additional caution and not as a standalone assay.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None. The test item tissue was considered viable (MTT uptake visual evaluation). The NC test tissues were similarly viable. The PC indicated blue/white tissue therefore only: semi-viable

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is (post study) validated. The test laboratory had previously demonstrated technical proficiency and this information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Although the absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC rather than later proscribed Ca2+/Mg2+-free DPBS. The NC was tested concurrently. The difference in viability was < 10% between replicates.
- Acceptance criteria met for positive control: No. Tissue variability was >20% in PC. The results of the assay should be interpreted with additional caution and not as a standalone assay.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information. See further comments above.

Interpretation of results:

other: Using the results of the study as a standalone assay: under the GHS criteria “no prediction can be made” ; the test item is suggested to have "limited" potential for eye irritation as part of weight of evidence.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, as a standalone assay: under the GHS criteria “no prediction can be made” ; the test item is suggested to have "limited" potential for eye irritation as part of weight of evidence. The test item had a relative mean viability was > 60% and therefore the test item was considered potentially not a serious eye irritant or corrosive. However, under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. Additionally, the 1%w/v SDS positive control, gave a mean viability 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Additionally, the test exposure was 10 minutes rather than the modern OECD TG 492 guideline specified 30 minutes. Therefore the results of the assay should be interpreted with additional caution and not as a standalone assay.

Executive summary:

The study was performed to assess the eye irritancy potential of the test item using the SkinEthic HCE EIT model in accordance with GLP. Duplicate tissues were treated with 30 µL of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Duplicate tissues were treated with 30 µL of solution A to serve as negative controls and duplicate tissues were treated with 30 µL of 1% w/v SDS to serve as positive controls. “Solution A" was used as supplied ; composition per litre : Na2HPO4 0.142 g/L; Glucose 1.802 g/L ; HEPES 7.149 g/L ; KCl 0.224 g/L ; NaCl 7.597 g/L. This solution was provided in accordance with the test kit manufacturer instructions. The plates were incubated at 37°C, 5% CO2 in air during the exposure time. Rinsing consisted of initially rinsing each tissue insert using DPBS. Rinsing was achieved by filling and emptying tissue Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µL of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air. At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader. In pre-testing the test item did not directly interact with MTT and therefore was a direct MTT reducer. The relative mean tissue viability obtained after 10 minutes treatment with the test item compared to the negative control tissues was 85%. The positive control had a mean cell viability of 39% after 10 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the test considered acceptable. Under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. The difference in viability was < 10% between replicates. The positive control gave a response within an expected range, mean = 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Under the conditions of this study, under the GHS criteria “no prediction can be made” ; the test item is suggested to have "limited" potential for serious eye irritation as part of weight of evidence. The test item had a relative mean viability was > 60% and therefore the test item was considered potentially not a serious eye irritant or corrosive. However, under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. Additionally, the 1%w/v SDS positive control, gave a mean viability 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Additionally, the test exposure was 10 minutes rather than the modern OECD TG 492 guideline specified 30 minutes. Therefore the results of the assay should be interpreted with additional caution and not as a standalone assay.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

Rabbit Enucleated Eye Test (REET) :completed prior to any in vivo testing in accordance with OECD TG 405

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-04-2010 to 11-06-2011

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:

Conducted equivalent to or similar to recognised guideline/protocol and performed under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

(2002) / Preliminary Test: Rabbit Enucleated Eye Test (REET) : this test was completed prior to in vivo testing in accordance with OECD TG 405 as a step-wise procedure in the interest of animal welfare. This study was completed to ascertain any ocular irritancy potential of the test item in the rabbit following application onto the cornea of the enucleated eye. The REET was completed to confirm information that the test item may cause irritation in a rabbit eye.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICCVAM-Recommended Test Method Protocol Isolated Rabbit Eye Test Method ; NIH Publication No. 10-7553 (2010)

Version / remarks:

ICCVAM-Recommended Test Method Protocol Isolated Rabbit Eye Test Method
Originally published as Appendix B5 of “ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products”
NIH Publication No. 10-7553 – Published 2010
Available at: http://iccvam.niehs.nih.gov/methods/ocutox/MildMod-TMER.htm

Deviations:

yes

Remarks:

no concurrent positive control was included in the assay

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2009 ; signature: November 2009

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): Not specified. For OECD TG 405 : 12 to 20 weeks and 2.0 to 3.5 kg is typical.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Donor rabbits were humanely euthanised on site. Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1% w/v). Examination was aided with the SL-5 slit-lamp biomicroscope. Corneal thickness values were also recorded using the DGH-1000 Ultrasonic pachymeter. Any eyes that showed evidence of ocular irritation or defect were excluded. Extraction time and placing eyes in the superfusion chamber following enucleation was minimized. Immediately following termination, two to three drops of saline solution (approximately 32°C) were applied to the cornea to prevent desiccation during excision. The eye was then carefully removed, positioned in a Perspex clamp and placed within the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber adjusted so that saline solution was allowed to irrigate the surface of the cornea. The eyes were then allowed to equilibrate for approximately thirty minutes. Following the equilibration period, the eyes were re-examined to ensure they had not been damaged during excision. Corneal thickness was also measured using the ultrasonic pachymeter. Any eyes in which the corneal swelling was greater than 10% relative to the pre-enucleation measurement, or in which the cornea was stained with fluorescein, were
rejected. The post equilibration corneal thickness values for each eye were recorded.
- Time interval prior to initiating testing: Same day (< 24 hours)
- indication of any existing defects or lesions in ocular tissue samples: No. Any eyes that showed evidence of ocular irritation or defect were excluded.
- Indication of any antibiotics used: No.

Vehicle:

unchanged (no vehicle)

Controls:

other: left eye remained untreated

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.

Duration of treatment / exposure:

The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline (0.9% sodium chloride)
Controls (negative and/or positive control items, where applicable) were similarly applied to the cornea in the negative and positive control groups (wherea applicable) respectively.

Observation period (in vivo):

Treated corneas were evaluated prior to treatment and at 60, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Number of animals or in vitro replicates:

Triplicate (n=3) for test item and reference item (where applicable) ; duplicate (n=2) for negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: Full details are provided in the full study report.

EQUILIBRATION AND BASELINE RECORDINGS: Taken at zero time after 30 minutes incubation prior to exposure.

NUMBER OF REPLICATES: Triplicate (3) for test item, positive control (where applicable) and duplicate (2) negative controls.

NEGATIVE CONTROL USED: sodium chloride solution 0.9% w/v

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED : Not applicable (no concurrent positive control)

APPLICATION DOSE AND EXPOSURE TIME: Application dose: 0.1 mL ; Exposure time: 10 seconds.

OBSERVATION PERIOD: prior to treatment and at 60, 120, 180 and 240 minutes (±5 minutes) after decontaminated with saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Test item remained in place for 10 seconds and then rinsed with 20 mL isotonic saline. Treated corneas were evaluated prior to treatment and at 60, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
- Indicate any deviation from test procedure in the Guideline: Not applicable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Microscope. Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment, according to the numerical evaluation given in Appendix 2 adopted from Advances in Modern Toxicology: Dermatoxicology, 4th Ed., (Marzulli and Maibach, editors) Hemisphere Publishing Corporation, USA (1991).
- Damage to epithelium based on fluorescein retention: Microscope. The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post
equilibration and approximately 240 minutes following treatment, according to the numerical evaluation (McDonald - Shadduck Score System)
- Swelling: measured with optical pachymeter on a slit-lamp microscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each
enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
- Macroscopic morphological damage to the surface: Yes. Observations for pitting, mottling or sloughing were made.
- Others (e.g, histopathology): Not applicable. Samples were taken for possible histopathology where relevant.

SCORING SYSTEM:
- Mean corneal swelling (%): See tables 1 to 6.
- Mean maximum opacity score: See tables 1 to 6.
- Mean fluorescein retention scores at 240 minutes post-treatment: See tables 1 to 6.

DECISION CRITERIA: The data for all endpoints was assessed and an estimate of the test item ocular irritancy potential was made based on the following cut-off values: REET parameter / Cut-off :
(i) Maximum Corneal Opacity (Corneal Cloudiness x Area) / > or = 4
(ii) Maximum Fluorescein Uptake (Intensity x Area) / > or = 4
(iii) Mean Corneal Swelling (mins): 60, 120, 240 / > or = 25%
(iv) Corneal Epithelium Observations / Any with pitting, mottling or sloughing
Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes). A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 minutes post treatment observation periods. Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant. Full details are provided in the full study report.
In this context, a positive would indicate the test item would be considered have the potential to meet GHS Eye Irritation Category 2 or 1 classification.

Irritation parameter:

cornea opacity score

Remarks:

240 minutes

Run / experiment:

mean (n=3)

Value:

3.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Remarks:

mean score = 3.33 (all eyes ; 240 min time point) ; 2/3 eyes had positive > or = 4 score ; some loss of transparency was noted in all test eyes at all observations.

Irritation parameter:

percent corneal swelling

Remarks:

corneal thickness / 240 minutes

Run / experiment:

mean (n=3)

Value:

76.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Remarks:

mean score = 76.8% (all eyes ; 240 min time point) ; 3/3 eyes had positive > or = 25% score

Irritation parameter:

morphological effects

Remarks:

Corneal Epithelium Condition / 240 minutes

Run / experiment:

mean (n=3)

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Remarks:

240 min time point ; 2/3 eyes had positive (sloughing observed) Corneal Epithelium Condition

Irritation parameter:

fluorescein retention score

Remarks:

240 minutes

Run / experiment:

mean (n=3)

Value:

6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Remarks:

240 min time point ; 2/3 eyes had positive (score 8) Maximum Fluorescein Uptake (intensity x area)

Table 1 : REET - Individual Scores for Corneal Opacity

	Test Eyes												Control Eyes
Chamber Number	1A				3A				5A				2A				4A
Time After Treatment (minutes)	60	120	180	240	60	120	180	240	60	120	180	240	60	120	180	240	60	120	180	240
Degree of Corneal Opacity	1	1	1	1	1	1	1	1	1	1	1	1	0	0	0	0	0	0	0	0
Area of Corneal Opacity	3	3	2	2	3	4	4	4	3	4	4	4	0	0	0	0	0	0	0	0
Maximum Corneal Opacity (corneal cloudiness x area)	3	3	2	2	3	4+	4+	4+	3	4+	4+	4+	0	0	0	0	0	0	0	0

+ = meets or exceeds cut-off value indicating severe ocular irritant

 

Table 2 : REET - Individual Measurements for Corneal Thickness (µm)

Test Eyes
Time After Treatment (minutes)		60						120						180						240
Corneal Position		oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean
Chamber Number	1A	418	409	408	429	412	415.2	437	422	423	442	434	431.6	439	427	424	446	457	438.6	441	444	423	445	490	448.6
	3A	426	418	408	450	415	423.4	580	456	497	600	452	517.0	683	603	531	669	521	601.4	750	764	630	788	742	734.8
	5A	445	420	449	448	426	437.6	553	484	512	576	460	517.0	634	553	573	732	639	626.2	650	583	653	752	769	681.4
Control Eyes
Time After Treatment (minutes)		60						120						180						240
Corneal Position		oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean	oc	1	2	3	4	mean
Chamber Number	2A	406	372	373	387	385	384.6	397	359	372	383	370	376.2	395	354	363	374	372	371.6	394	349	362	362	360	365.4
	4A	448	411	422	436	418	427.0	437	403	409	427	412	417.6	431	398	410	421	403	412.6	422	399	396	412	391	404.0

oc= Optical centre

mean = Mean corneal thickness

 

Table 3 : REET - Determination of Corneal Swelling (percentage) Test Item Group

Test Eyes
Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a
1A	Post equilibration	353.0	N/A	3A	Post equilibration	356.2	N/A	5A	Post equilibration	345.8	N/A
	60 Post treatment	415.2	17.6		60 Post treatment	423.4	18.9		60 Post treatment	437.6	26.5
	120 Post treatment	431.6	22.3		120 Post treatment	517.0	45.1		120 Post treatment	517.0	49.5
	180 Post treatment	438.6	24.2		180 Post treatment	601.4	68.8		180 Post treatment	626.2	81.1
	240 Post treatment	448.6	27.1		240 Post treatment	734.8	106.3		240 Post treatment	681.4	97.1

N/A = not applicable

+ = Meets or exceeds cut-off value and therefore indicates a severe eye irritant

 

Test Eyes

Mean corneal swelling 1 hour following treatment 21.0%

Mean corneal swelling 2 hours following treatment 39.0%+

Mean corneal swelling 4 hours following treatment 76.8%+

 

Table 4 : REET - Determination of Corneal Swelling (percentage) Control Group

Control Eyes
Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a	Chamber Number	Observation Period (minutes)	Mean Corneal Thickness (µm)	Corneal Swelling (%)a
2A	Post equilibration	344.4	N/A	34A	Post equilibration	394.8	N/A
	60 Post treatment	384.6	11.7		60 Post treatment	427.0	8.2
	120 Post treatment	376.2	9.2		120 Post treatment	417.6	5.8
	180 Post treatment	371.6	7.9		180 Post treatment	412.6	4.5
	240 Post treatment	365.4	6.1		240 Post treatment	404.6	2.3

Control Eyes

Mean corneal swelling 1 hour following treatment 9.9% Mean corneal swelling 2 hours following treatment 7.5%

Mean corneal swelling 4 hours following treatment 4.2%

 

Table 5 : REET - Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

	Test Eyes			Control Eyes
Chamber Number	1A	3A	5A	2A	4A
Intensity of Fluorescein Uptake	1	2	2	0	0
Area of Fluorescein Uptake	2	4	4	0	0
Maximum Fluorescein Uptake (intensity x area)	2	8+	8+	0	0

+ = Meets or exceeds cut-off value and therefore indicates a severe eye irritant

 

Table 6 : REET - Corneal Epithelium Condition

Test Eyes
Chamber Number	Time After Treatment (minutes)
	60	120	180	240
1A	Normal	Normal	Normal	Normal
3A	Normal	Sloughing+	Sloughing+	Sloughing+
5A	Normal	Sloughing+	Sloughing+	Sloughing+
Control Eyes
Chamber Number	Time After Treatment (minutes)
	60	120	180	240
2A	Normal	Normal	Normal	Normal
4A	Normal	Normal	Normal	Normal

+ = Meets or exceeds cut-off value and therefore indicates a severe eye irritant

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item has the potential to meet GHS Eye Irritation category 2 or 1. The overall evidence will be assessed in weight of evidence determination. The weight of evidence (from available eye irritation in vitro assays) that the test item has the potential to induce GHS Eye Irritation Category 2.

Executive summary:

The study was performed in a step-wise manner according to OECD TG 405 under GLP. Prior to testing in vivo, the Rabbit Enucleated Eye Test (REET) in vitro test was conducted to assess the ocular irritancy potential of the test item in the rabbit following application onto the cornea of the enucleated eye. 0.1 mL of the test item was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32°C ± 1.5°C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride). The maximal ocular irritation scores were as follows: corneal opacity score: 4, fluorescein uptake 8, corneal swelling at 60, 120, 240 minutes: 21.0, 39.0 and 76.8% respectively in test item group versus 9.9, 7.5 and 4.2%, respectively in the (negative) control group. In the test item group there was evidence of sloughing of the corneal epithelium. Following assessment of the data for all endpoints the test item was considered to have the potential to cause severe ocular irritancy in vivo. The in vivo eye irritation study is therefore not required. Applicant assessment indicates, under the conditions of this study the test item has the potential to meet GHS Eye Irritation category 2 or 1. The overall evidence will be assessed in weight of evidence determination.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Irritation:

Key study : in vivo, OECD TG 404, 2010 : The study was performed to OECD TG 404 and to assess the primary skin irritancy potential of the test item in New Zealand White rabbits in accordance with GLP. The test item was applied to two males f by semi-occluded application to the intact rabbit skin with 0.5 mL test item introduced under a 2.5 cm x 2.5 cm cotton gauze patch on the clipped skin or 4-hours. The patch was secured in position with a strip of surgical adhesive tape. After exposure to the test item, the patches were removed and individual dose sites were scored at approximately 1, 24, 48, and 72 hours. No corrosive effects were noted. Well-defined erythema (score =2) and slight oedema (score = 2) were noted at both (n=2) treated skin sites at the 24, 48 and 72-Hour observations. Other skin reactions noted at both treated skin sites at the 48 and 72-Hour observations were light brown discolouration of the epidermis and loss of skin flexibility and/or elasticity. Crust formation, prevented accurate evaluation of erythema and oedema, was noted at both treated skin sites at the 7-Day observation. Mean scores following grading at 24, 48 and 72h were score 2.0/2.0 in erythema and eschar and score 2.0/2.0 in oedema scoring criteria, respectively. Slight desquamation was noted at both treated skin sites at the 14-Day observation. Under the conditions of the study, the test item is considered to be a skin irritant.

Applicant assessment indicates: that the CLP Regulation (EC) 1272/2008 Skin Irritation : category 2 classification within the inflammation criteria (erythema/eschar and oedema) being mean score at 24, 48 and 72 h > or = 2.3 in two (of three potential) sites was not met. The study was originally classified under DSD 67/548/EEC which utilised a mean score of 2.0 for positive result. The study was terminated on day 14 under DSD 67/548/EEC, all erythema/eschar and oedema inflammation had ceased although ‘slight desquamation’ persisted in two (of three potential) sites to day 14. Therefore, CLP Regulation (EC) 1272/2008 : Skin Irritant Category 2 classification is maintained.

Key study : in vitro, EU Method B.46, 2010 : The study was performed to EU Method B.46 and/or a method equivalent to OECD TG 439 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (EPISKIN Small Model). In pretesting, the solution containing the test item was colourless and therefore there were no indications of colour interference in the report. It was therefore appeared unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: the MTT solution containing the test item turned blue or purple which indicated that the test item did have the potential directly reduce MTT. It was necessary to run killed tissues and correction as part of the test. This was done by addition to the normal test procedure the MTT reducing test material was applied to one water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed tissue demonstrates a small amount of MTT reduction due to residual reducing enzymes. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The calculation was specified as: Corrected viability of treated killed tissues = 0.116 (tkt) - 0.066 (ukt) = 0.050, and where : tvt = treated viable tissues ; tkt = treated killed tissues and/or ukt = untreated killed tissues, respectively. The relative mean viability of the test item treated tissues was 50.5% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 20%. The limit was 20% at the time of the draft OECD guideline (2020). < 18% was not achieved. However the test item was classified as Skin Irritant and therefore this requirement does not need to be strictly applied in interpreting the results. The mean OD540 for the negative control treated tissues was 0.877 and the standard deviation value of the viability was 6.7%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 4.7% relative to the negative control treated tissues and the standard deviation value of the viability was 17.5%. The positive control acceptance criteria were therefore satisfied. Under the conditions of this study, the test item is considered to be (borderline) irritating to the skin.

 

Eye Irritation:

Key study : in vitro, SkinEthic ECE EIT, 2010 : The study was performed to assess the eye irritancy potential of the test item using the SkinEthic HCE EIT model in accordance with GLP. Duplicate tissues were treated with 30 µL of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Duplicate tissues were treated with 30 µL of solution A to serve as negative controls and duplicate tissues were treated with 30 µL of 1% w/v SDS to serve as positive controls. “Solution A" was used as supplied ; composition per litre : Na2HPO4 0.142 g/L; Glucose 1.802 g/L ; HEPES 7.149 g/L ; KCl 0.224 g/L ; NaCl 7.597 g/L. This solution was provided in accordance with the test kit manufacturer instructions. The plates were incubated at 37°C, 5% CO2 in air during the exposure time. Rinsing consisted of initially rinsing each tissue insert using DPBS. Rinsing was achieved by filling and emptying tissue Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µL of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air. At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader. In pre-testing the test item did not directly interact with MTT and therefore was a direct MTT reducer. The relative mean tissue viability obtained after 10 minutes treatment with the test item compared to the negative control tissues was 85%. The positive control had a mean cell viability of 39% after 10 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the test considered acceptable. Under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. The difference in viability was < 10% between replicates. The positive control gave a response within an expected range, mean = 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Under the conditions of this study, under the GHS criteria “no prediction can be made” ; the test item is suggested to have "limited" potential for serious eye irritation as part of weight of evidence. The test item had a relative mean viability was > 60% and therefore the test item was considered potentially not a serious eye irritant or corrosive. However, under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. Additionally, the 1%w/v SDS positive control, gave a mean viability 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Additionally, the test exposure was 10 minutes rather than the modern OECD TG 492 guideline specified 30 minutes. Therefore the results of the assay should be interpreted with additional caution and not as a standalone assay.

Key study : Enucleated Rabbit Eye Test (REET) - pretest screening to OECD TG 405, 2010 : The study was performed in a step-wise manner according to OECD TG 405 under GLP. Prior to testing in vivo, the Rabbit Enucleated Eye Test (REET) in vitro test was conducted to assess the ocular irritancy potential of the test item in the rabbit following application onto the cornea of the enucleated eye. 0.1 mL of the test item was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32°C ± 1.5°C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride). The maximal ocular irritation scores were as follows: corneal opacity score: 4, fluorescein uptake 8, corneal swelling at 60, 120, 240 minutes: 21.0, 39.0 and 76.8% respectively in test item group versus 9.9, 7.5 and 4.2%, respectively in the (negative) control group. In the test item group there was evidence of sloughing of the corneal epithelium. Following assessment of the data for all endpoints the test item was considered to have the potential to cause severe ocular irritancy in vivo. The in vivo eye irritation study is therefore not required. Applicant assessment indicates, under the conditions of this study the test item has the potential to meet GHS Eye Irritation category 2 or 1. The overall evidence will be assessed in weight of evidence determination.

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient irritating effects to the eye which are sufficient for classification based on the available: (i) SkinEthic HCE EIT assay (eq. or similar to OECD TG 492) but which could not be utilised as a standalone assay due to deviations to the modern protocol, indicating not eye irritating and/or (ii) the REET assay (pre-test to an OECD TG 405 study) predicting potential severe irritation but without concurrent positive controls to judge the sensitivity of the test system. There is adequate evidence to classify as GHS and/or CLP Regulation (EC) 1272/2008 : Eye Irritant : category 2. The substance possesses properties which are indicative of transient/reversible irritating effects on the eye sufficient for classification.

Respiratory Irritation:

INHALATION:

Key study : OECD TG 436, 2012 : The study was performed according to OECD TG 436 and EU Method B.52 in accordance with GLP to assess the acute inhalation toxicity of the test item. Following a sighting test at 2.22 mg/L mean achieved concentration, one group of six RccHanTM : WIST strain rats (three males and three females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fourteen-day observation period. The mean achieved atmosphere concentrations was Group 1: 0.98 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction < 4 μm: Group 1: 1.64 μm and 83.8%. The Geometric Standard Deviation was 2.49. There was no mortalities in Group 1. Common abnormalities noted during the study included decreased respiratory rate, ataxia, hunched posture, pilo-erection and wet fur. Frequent instances of increased respiratory rate and an isolated occurrence of laboured respiration were also noted. Animals recovered to appear normal on Day 4 post-exposure. All males/females exhibited slight bodyweight losses on the first day postexposure. Reasonable bodyweight development was noted in all animals during the remainder of the recovery period with the exception of one female animal which exhibited a slight bodyweight loss from Days 3 to 7 post-exposure, but which was gaining bodyweight at from Days 7 to 14 (+3 g). Two males (#1 and #3) and one female (#2) showed no abnormalities. Dark and/or pale patches on the lungs were noted amongst one male (#2) and two of three females (#1 and #3). As the mean achieved concentration was 98% of target and no deaths occurred, no further levels were required as it was considered that at the next dose level of 5 mg/L none of the males/females were considered to survive. This is further confirmed by the observations and the death noted at a mean achieved atmosphere concentration of 2.22 mg/L during the sighting study. Under the conditions of this study, the 4-hour inhalation LC50 (male/female) was considered to be > 1.0 and ≤ 5.0 mg/L within the RCCHan WIST rat.

 

Applicant assessment indicates, although the lungs indicated post-necropsy findings (dark patches), there was no mortality, relevant clinical signs and no functional impairment. All males/females were gaining bodyweight at the end of the study. There was additionally no indications during necropsy of findings in the upper respiratory tract which was subject to a detailed macroscopic examination for signs of irritancy or local toxicity.

 

References:

1. OECD TG 436 (2009)

2. OECD TG 403 (2009)

2. OECD 39 (2009)

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for dermal irritation category 2: H315

The substance meets classification criteria under Regulation (EC) No 1272/2008 for eye irritation category 2: H319

 

For skin irritation, further in vitro skin corrosion testing does not need to be conducted based on the available information allowing a definitive conclusion on the classification of the substance. The substance does not demonstrate sufficient skin corrosion potential necessary for classification and labelling. The substance was classified within an available skin irritation in vivo assay (OECD TG 404) for skin irritation on a precautionary basis. Applicant assessment indicates: that the CLP Regulation (EC) 1272/2008 Skin Irritation : category 2 classification within the inflammation criteria (erythema/eschar and oedema) being mean score at 24, 48 and 72 h > 2.3 in two of three sites was not met. The study was originally classified under DSD 67/548/EEC which utilised a mean score of 2.0 for positive result. The study was terminated on day 7 under DSD 67/548/EEC, all inflammation had ceased although ‘slight desquamation’ persisted in two of three sites to day 7. The conclusion is also considering the preceding available in vitro skin irritation assay (EU Method B.46) that was borderline irritant at relative viability 50.5%. This was considering the generally deviations compared to the modern OECD guideline (2020) and the totality of the information.

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient irritating effects to the eye which are sufficient for classification based on the available: (i) SkinEthic HCE EIT assay (eq. or similar to OECD TG 492) but which could not be utilised as a standalone assay due to deviations to the modern protocol, indicating not eye irritating and/or (ii) the REET assay (pre-test to an OECD TG 405 study) predicting potential severe irritation but without concurrent positive controls to judge the sensitivity of the test system. There is adequate evidence to classify as GHS and/or CLP Regulation (EC) 1272/2008 : Eye Irritant : category 2. The substance possesses properties which are indicative of transient/reversible irritating effects on the eye sufficient for classification.

 

Within the respiratory route, there were some indications of sensory irritation, however, there was an absence of other relevant clinical signs and/or macroscopic correlates and/or body weight declines that persisted during the study period. As a consequence, the substance is not classified for respiratory irritation or local cytotoxic effects in the respiratory tract. Additionally, separate to the above the substance is classified under GHS Acute Toxicity Inhalation: category 4.

 

References:

1. Guidance on Application of the CLP Criteria, ECHA, version 5.0, July 2017