dec-1-en-4-yne

Endpoint summary

Description of key information

Skin irritation: non-irritating (60 minute/42 hour: relative mean viability = 87.6%), OECD TG 439, 2018

Skin irritation: non-irritating, OECD TG 404, 2018

Eye irritation, in vitro: non-irritating, mean IVIS = 2.09 and mean permeability = 0.005, OECD TG 437, 2018

Eye irritation: non-irritating, OECD TG 405, 2018

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:

30-05-2017 to 30-06-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2015 ; signature: September 2015

Test system:

human skin model

Remarks:

EPISKIN TM Small Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM TM Epi-200- SIT
- Tissue batch number(s): Lot no.: 25825
- Production date: Not reported.
- Shipping date: Not reported.
- Delivery date: 27-06-2017 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 27-05-2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C, 5 ± 0.5% CO2 ; Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation medium will be changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approximately 41 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test item. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate for incubation.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution prepared in assay medium
- Incubation time: The plate was post-42 hour incubation period – further incubated (37 ± 1.5 °C, 5 ± 0.5% CO2) for 3 hours for the MTT assay.
- Spectrophotometer: microplate reader
- Wavelength: 570 nm (OD570)
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60-minute exposure/42-hour incubation period. With standard deviation of 0.6%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.670 and the standard deviation of viability was 4.6%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 7.0%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Three (3), triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 60-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 60-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 60-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference was completed.

OTHER:
Epi-200- SIT Model (Lot no.: 25825). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅). The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential. As a complimentary endpoint the concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that 1. air bubbles between agarose and insert were not > 30% of the total surface, 2. liquid on top of the insert was removed with sterile cotton tips, 3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded. 0.9 mL of the assay medium (20 – 25 °C) were pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 22 hours (37 ± 1.5 °C, 5 ± 0.5% CO2, 95 ± 5% RH).

Application of test item and rinsing:
30 μL (47 μL/cm2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue for a complete treatment time of 60 minutes. The test was performed under monochromatic yellow light. After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test item. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate for incubation. Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The tissues were subsequently placed into was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium (and sample stored in the freezer). After incubation medium will be changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approximately 41 hours. Following ca. 42 hour post exposure incubation the treated plates were then tested for MTT formazan extraction.

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

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.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl ; Dulbecco’s Phosphate Buffered Saline (DPBS)
- Concentration (if solution): Tested as supplied (undiluted).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl ; The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution.
- Concentration (if solution): 5%

Duration of treatment / exposure:

Tissues were treated with the test item for 60 minutes and then washed with DPBS to remove residual test item. Negative control and/or Positive control were similarly treated with the respective reference items (DPBS and/or SDS 5%).

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C. Then 3 hours incubation prior to and/or MTT loading and Formazan extraction.

Number of replicates:

Triplicate (n=3) ; treatment and concurrent negative control and positive control groups

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

87.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 minute exposure. Reversibility: no data. Remarks: n=3; SD = 7.0% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has previously demonstrated technical proficiency. Also see historic control data.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Dose Group	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 3 Tissues	Rel. Absorbance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [%] or “Mean viability”
Blank		0.038	0.038	0.037	0.037	0.000
Negative Control	1	1.708	1.718	1.740	1.722	1.684	1.670	100.8	4.6	100.0
	2	1.804	1.757	1.767	1.776	1.738		104.1
	3	1.659	1.606	1.611	1.625	1.588		95.1
Positive Control	1	0.090	0.095	0.094	0.093	0.055	0.055	3.3	0.6	3.3
	2	0.093	0.093	0.091	0.092	0.055		3.3
	3	0.092	0.093	0.093	0.093	0.055		3.3
Test Item	1	1.406	1.385	1.369	1.387	1.349	1.464	80.8	7.0	87.6
	2	1.552	1.532	1.523	1.536	1.498		89.7
	3	1.590	1.562	1.593	1.582	1.544		92.5

 

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues (mean OD: between 1.625 and 1.776). Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.3% (= ≤ 20%) thus ensuring the validity of the test system. The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were 7%.All assay acceptance criteria were met.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this in vitro study, the test item is not considered to be irritating to skin.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (Epi-200- SIT / EPIDERM Model). Triplicate tissues were treated with the test item, negative control and positive control items for an exposure period of 60 minutes. The test item, the negative control (DPBS) and of the positive control (5% SLS) were applied to each triplicate tissue, and spread to match the surface of triplicate tissue. At the end of the exposure period each tissue was rinsed before incubating for ca. 42 hours. After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1.5 °C, 5 ± 0.5 % CO2), the tissues were rinsed three times with DPBS, and carefully dried with blotting paper. The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 24-well plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for 2.5 hours while shaking at room temperature. After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour. Per each tissue, three individual 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 87.6% after the 60-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 7.0%. The mean OD570 for the negative control treated tissues was 1.670 and the standard deviation value of the viability was 4.6%. The absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-08-2017 to 27-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2017 ; signature: November 2017

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

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 - 52 weeks
- Weight at study initiation: 3.16 to 4.09 kg
- Housing: Individually housed in suspended cages.
- Diet: certified rabbit diet ad libitum
- Water: mains drinking water ad libitum
- Acclimation period: at least 5 days

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

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5mL
- Concentration (if solution): Test material was used as supplied.

Duration of treatment / exposure:

4 hours

Observation period:

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

Number of animals:

3 ; following the guideline sequential testing approach

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

OBSERVATION TIME POINTS
(indicate if minutes, hours or days): immediately, 1 hour, then 24/48/72 hours, with 7-day and 14-day observations (as appropriate)

SCORING SYSTEM:
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:

animal #1

Time point:

other: 1h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 1h

Score:

2

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 1h

Score:

1

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: mean of 24 and 72 h observation due to technical error

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks:

score 0 (fully reversed) but slight desquamation at day 14

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 1h

Score:

0

Max. score:

4

Remarks on result:

other: mean of 24 and 72 h observation due to technical error

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 1h

Score:

1

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 1h

Score:

2

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks:

score 0 (fully reversed) but slight desquamation at day 14

Irritant / corrosive response data:

- Erythema: See tables
- Edema: See tables.
- Reversibility of effects: All effects had ceased in 2 of 3 females at day 14. All rabbits had score = 0 at the end of the observation. Slight desquamation was observed in 1 of 3 females.

Other effects:

- Other adverse local effects: None reported. See tables above. All effects had ceased in 2 of 3 females at day 14. All rabbits had score = 0 at the end of the observation. Slight desquamation was observed in 1 of 3 females.
- Other adverse systemic effects: Two females (#2 and #3) gained bodyweight during the study (+0.08 to +0.10 g) and one females had a body weight loss (-0.10 g). Applicant assessment indicates that the bodyweight loss was very minor and there were no clinical sign correlations reported.

Table 1. Individual skin reactions

Skin Reaction	Observation Time	Individual Scores
		1 (female)	2 (female)	3 (female)
Erythema/Eschar formation	1 hour	0	2	1
	24 hours	0	2 Br	1
	48 hours	X	2 Br	1
	72 hours	0	2 BrLe	1 Le
	7 days	-	2 Cf	1 LeCf
	14 days	-	0	0 DG
Oedema formation	1 hour	0	1	2
	24 hours	0	2	2
	48 hours	X	2	1
	72 hours	0	1	1
	7 days	-	1	1
	14 days	-	0	0

Br = Light brown discoloration of the epidermis

Le = Loss of skin elasticity

Cf = Crust formation

G = Glossy skin

D = Slight desquamation

x = observation not performed due to technician error

- = observation not required

 

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

Erythemea/Escar Formation:

1: total = 0 ; mean score = 0.0 (note mean of 24 and 72 h observations)

2: total = 6 ; mean score = 2.0 (persisted to 7 days, reversed at day 14)

3: total = 3 ; mean score = 1.0 (score 0 and slight desquamation at day 14)

Oedema Formation:

1: total = 0 ; mean score = 0.0 (note mean of 24 and 72 h observations)

2. total = 5 ; mean score = 1.67 (persisted to 7 days, reversed at day 14)

3. total = 4 ; mean score = 1.33 (score 0 and slight desquamation at day 14)

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item is not considered to be irritating.

Executive summary:

The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 to assess the primary skin irritancy potential of the test substance in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 mL of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored immediately and at approximately 1, 24, 48, and 72 hours. Three females were exposed following the guideline sequential testing approach. A single 4-Hour, semi occluded application of the test item to the intact skin of three rabbits produced very slight (score 1) to well-defined erythema (score 2) and very slight (score 1) to slight edema (score 2) at two treated skin sites. Light brown discoloration of the epidermis, loss of skin elasticity, crust formation, slight desquamation and glossy skin were also noted at these two treated skin sites. No evidence of skin irritation was noted at one treated skin site. No corrosive effects were noted. Mean scores for following grading at 24, 48 and 72h were 0.0, 2.0 and 1.0 in erythema and eschar and 0.0, 1.67 and 1.33 in edema scoring criteria. All effects had ceased in 2 of 3 females at day 14. All rabbits had score = 0 at the end of the observation. Slight desquamation was observed in 1 of 3 females. Under the conditions of the study, the test item is not considered to be a skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-07-2017 to 02-03-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2015 ; signature: September 2015

Species:

other: bovine

Strain:

not specified

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): > 9 months old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
Post-excision, placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics over ice packs.
- Time interval prior to initiating testing: < 24 hours. Corneas were prepared for testing immediately on same day arrival.
- indication of any existing defects or lesions in ocular tissue samples: None. Only corneas free from damage utilised (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches were discarded).
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL during transport.

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): undiluted

Duration of treatment / exposure:

10 minutes at 32 ± 1ºC.

Duration of post- treatment incubation (in vitro):

The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. A post-treatment opacity reading was taken and each cornea was visually observed (t130).

Number of animals or in vitro replicates:

Three (3) per test item, or negative or positive controls, respectively.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with Incubation medium. The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 μg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS). The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that 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. At the pre-exposure incubation period, the basal opacity was determined (t0).

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches) and where necessary discarded. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

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

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: 2-ethoxyethanol; 99.0% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes at 32 ± 1ºC.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: 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. At the pre-exposure incubation period, the basal opacity was determined (t0).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber. After the test item or control items, respectively, were rinsed off from the application side with saline, fresh complete medium (cMEM) was added into the anterior compartment.

- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. A post-treatment opacity reading was taken and each cornea was visually observed (t130).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: Opacity, Permeability and In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD492) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD492 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (n=3)

Value:

2.09

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. The corneas treated with the test item did not cause a relevant increase of the corneal opacity. For corneas treated with the negative control item neither an increase of opacity nor permeability could be observed. The corneas treated with the positive control item was tested undiluted and showed clear opacity and distinctive permeability of the corneae.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test facility was a validated laboratory (information in the public domain) and/or concurrent positive and negative controls were within acceptable limits.

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:
1. 2-ethoxyethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean of for the test facility. This criterion was met (actual mean IVIS = 92.17)
2. sodium chloride 0.9% w/v solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). This criterion was met (actual mean opacity = 0.00 ; permeability = 0.059 and IVIS = 0.88)
Full details of the HCD is provided in the full study report.

 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity	Permeability (OD490)	In Vitro Irritancy Score
		Post-Incubation - Pre‑Treatment
Negative Control	1	0	0.055*	0.83
	2	0	0.064*	0.96
	3	0	0.057*	0.86
	Mean	0.00	0.059*	0.88
Positive Control	4	78.00*	0.894*	91.42
	5	75.00*	0.849*	87.74
	6	85.00*	0.774*	97.37
	Mean	-	-	92.17
Test Item	7	2.00*	0.003*	2.05*
	8	2.00*	0.009*	2.14*
	9	2.00*	0.004*	2.07*
	Mean	-	-	2.09

OD = Optical Density

* = corrected values

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be irritant in the in vitro eye corrosion/irritation test using Bovine Corneal Opacity and Permeability model. The in vitro irritancy score (IVIS) was < 3.0 in the prediction model.

Executive summary:

The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the test material in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test item was tested through topical application for 10 ± 1 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (2-ethoxyethanol), was 92.17 (GHS Category 1 eye damage prediction) and was within the historical positive control data range. The test item did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score (IVIS) of 2.09 after 10 minutes of treatment. Since the IVIS was < 3.0 the test item was predicated as not irritating to the eye. Under the conditions of this study the test item is not considered to be an irritant or corrosive to the eye in the Bovine Corneal Opacity and Permeability test.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-09-2017 to 16-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2017 ; signature: November 2017

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (documented in the full study report)
- Age at study initiation: 12-52 weeks old
- Weight at study initiation: 3.70 – 4.68 kg
- Housing: individually housed in suspended metal cages; with environment enrichment
- Diet: certified rabbit food ad libitum
- Water: mains water ad libitum
- Acclimation period: at least 5 days

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

IN-LIFE DATES: 05-09-2017 to 16-09-2017

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

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

Duration of treatment / exposure:

A volume of 0.1 mL of the test item, was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item and then released. The left eye remained untreated and was used for control purposes.

Observation period (in vivo):

Ocular assessment was conducted at approximately 1, 24, 48 and 72 hours after instillation of the test substance, according to numerical evaluation. Additional observation at day 7 and day 14 (as appropriate).

Number of animals or in vitro replicates:

2 (female). Testing was conducted sequentially. The response in those animals was such that exposure of a third would not affect classification of the test item, therefore, no further testing was needed under guidelines.

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit. The other animals were treated in a similar manner after considering the degree of eye irritation observed in the first and/or second animal.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM:
The irritation was assessed according to Draize (1977) numerical scoring system. At each observation period, the highest scores given were recorded. Any other ocular effects were also noted.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: mean; n=2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

other: mean; n=2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: mean; n=2

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.5

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: mean; n=2

Irritant / corrosive response data:

No corneal effects were noted. No iridial inflammation was noted. Moderate conjunctival irritation was noted in treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48 hour observations. Minimal conjunctival irritation persisted in one treated eye at the 72-Hour observation. All treated eyes appeared normal at the 72 hours to 7 days.

Other effects:

- Lesions and clinical observations: None reported.
- Ophthalmoscopic findings: Not applicable.
- Histopathological findings: Not applicable.
- Effects of rinsing or washing: Not applicable.
- Other observations: All females gained bodyweight during the study.

Table 1. Individual scores and mean scores for 24, 48 and 72 hours

Organism number	1					2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 days	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
Degree of Opacity	0	0	0	0	0	0	0	0	0
Mean (24 – 72 h)				0.0					0.0
IRIS	0	0	0	0	0	1	1	0	0
Mean (24 – 72 h)				0.0					0.3
CONJUNCTIVAE
Redness	2	2	1	1	0	2	1	1	0
Mean (24 – 72 h)				1.33					0.7
Chemosis	1	1	1	0	0	1	1	0	0
Mean (24 – 72 h)				0.7					0.3

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not irritating to the eye.

Executive summary:

The study was performed to OECD TG 405 and EU Method B.5 to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 mL of the test item was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Further observation was made at 7-days. A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Treated eyes appeared normal at the 72-Hour or 7-Day observations. No corneal or iridial effects were noted during the observation period. The calculated mean values based on the results from the 24, 48 and 72 hour readings were calculated as: cornea: 0; iris lesion: 0, 0.00 ; conjunctival redness: 1.33 and 0.67. Chemosis was 0.67, 0.33. 7 days after dosing all effects had fully reversed. Under the conditions of this study, the test item is not considered to be irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

Key study : In vitro, OECD TG 439, 2018 : The study was performed to OECD TG 439 and EU Method B.46 under GLP to assess the skin irritation potential of the test item using a human three dimensional epidermal model (Epi-200- SIT / EPIDERM Model). Triplicate tissues were treated with the test item, negative control and positive control items for an exposure period of 60 minutes. The test item, the negative control (DPBS) and of the positive control (5% SLS) were applied to each triplicate tissue, and spread to match the surface of triplicate tissue. At the end of the exposure period each tissue was rinsed before incubating for ca. 42 hours. After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1.5 °C, 5 ± 0.5 % CO2), the tissues were rinsed three times with DPBS, and carefully dried with blotting paper. The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 24-well plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for 2.5 hours while shaking at room temperature. After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour. Per each tissue, three individual 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 87.6% after the 60-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 7.0%. The mean OD570 for the negative control treated tissues was 1.670 and the standard deviation value of the viability was 4.6%. The absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6%. The positive control acceptance criteria were therefore satisfied. All acceptance criteria were considered to be met. Under the conditions of this study, the test item is not considered to be irritating to the skin.

 

Key study : In vivo, OECD TG 404, 2018 : The study was performed to OECD TG 404, EU Method B.4 and the Japanese MAFF (2000) and US EPA OPPTS 870.2500 to assess the primary skin irritancy potential of the test substance in accordance with GLP in New Zealand White rabbits. Following single 4-Hour, semi-occluded applications to the intact rabbit skin. 0.5 mL of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the clipped skin to assess the irritancy potential of the test item. The patch was secured in position with a strip of surgical adhesive tape. After 4 hours of exposure to the test item, the patches were removed and individual dose sites were scored immediately and at approximately 1, 24, 48, and 72 hours. Three females were exposed following the guideline sequential testing approach. A single 4-Hour, semi occluded application of the test item to the intact skin of three rabbits produced very slight (score 1) to well-defined erythema (score 2) and very slight (score 1) to slight edema (score 2) at two treated skin sites. Light brown discoloration of the epidermis, loss of skin elasticity, crust formation, slight desquamation and glossy skin were also noted at these two treated skin sites. No evidence of skin irritation was noted at one treated skin site. No corrosive effects were noted. Mean scores for following grading at 24, 48 and 72h were 0.0, 2.0 and 1.0 in erythema and eschar and 0.0, 1.67 and 1.33 in edema scoring criteria. All effects had ceased in 2 of 3 females at day 14. All rabbits had score = 0 at the end of the observation. Slight desquamation was observed in 1 of 3 females. Under the conditions of the study, the test item is not considered to be a skin irritant. Applicant assessment indicates: based on the presented mean scores and reversibility of effects the substance is considered to meet the criteria for GHS Skin Irritation: category 3, causes mild skin irritation. This criteria is not implemented within the Regulation (EC) 1272/2008 criteria.

 

Eye Irritation:

Key study : In vitro, OECD TG 438, 2018 : The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the test material in isolated bovine corneas. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The ocular irritancy of the test item was tested through topical application for 10 ± 1 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (2-ethoxyethanol), was 92.17 (GHS Category 1 eye damage prediction) and was within the historical positive control data range. The test item did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score (IVIS) of 2.09 after 10 minutes of treatment. Since the IVIS was < 3.0 the test item was predicated as not irritating to the eye. Under the conditions of this study the test item is not considered to be an irritant or corrosive to the eye in the Bovine Corneal Opacity and Permeability test.

Key study : In vivo, OECD TG 405, 2018 : The study was performed to OECD TG 405 and EU Method B.5 to assess the irritancy potential of the test item to the eye following a single application in the New Zealand White rabbit. A volume of 0.1 mL of the test item was placed into the conjunctival sac of one eye of two animals. The other eye remained untreated and was used for control purposes. The test was conducted in a stepwise manner conducted singularly. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Further observation was made at 7-days. A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Treated eyes appeared normal at the 72-Hour or 7-Day observations. No corneal or iridial effects were noted during the observation period. The calculated mean values based on the results from the 24, 48 and 72 hour readings were calculated as: cornea: 0; iris lesion: 0, 0.00 ; conjunctival redness: 1.33 and 0.67. Chemosis was 0.67, 0.33. 7 days after dosing all effects had fully reversed. Under the conditions of this study, the test item is not considered to be irritating to the eye.

Respiratory Irritation:

Key study : OECD TG 403, 2018 : The study was performed according to OECD TG 403 and EU Method B.2 in accordance with GLP to assess the acute inhalation toxicity of the test item. As insufficient data was available on the expected inhalation toxicity of the test item, a sighting test was performed to determine the initial exposure concentration. A group of one male and one female was exposed to an aerosol atmosphere of the test item at a target concentration of 3.5 mg/L (mean achieved concentration: 4.83 m/L). Based on the results of the sighting test, a limit test was performed. A group of ten animals (five males and five females) was exposed to an aerosol atmosphere of the test item at a target concentration of 5.0 mg/L for 4 hours using a nose only exposure system, followed by a fourteen day observation period. The mean achieved atmosphere concentrations was 4.90 mg/L The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: sighting test: 2.75 μm and 65.8% and Group 1 (4.90 mg/L limit test): 3.60 μm and 53.4%. The Geometric Standard Deviation was sighting test : 2.52 and Group 1 (4.90 mg/L limit test): 3.48, respectively. There were no male or female mortalities. In the definitive limit test - Group 1: Common abnormalities noted during the study included decreased respiratory rate, hunched posture, pilo-erection, body tremors and wet fur. There were frequent instances of ataxia and lethargy, occasional instances of areas of red/brown staining of the fur and fur stained yellow by test item and isolated instances of vocalization, prostration and areas of red/brown staining around the eyes. Animals recovered so that no significant abnormalities were apparent from Days 2 to 4 post-exposure. All animals exhibited body weight losses or no gain in body weight on the first day post-exposure. With the exception of two males and four females which exhibited body weight losses from Days 1 to 3 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period. All males/females were gaining bodyweight at the end of the recovery period. During necropsy in Group 1,: the following macroscopic abnormalities were detected : Lungs – Pale, abnormally red, dark patches ; Liver – Pale, patchy pallor (1 female) ; Kidneys – Pale (1 male) ; Large intestine – Gaseous distension (2 animals ; 1 male, 1 female). Under the conditions of this study, the inhalation LC50 (male/female) was > 4.90 mg/L within the RCCHan WIST rat. Applicant assessment indicates, although the lungs indicated post-necropsy findings, 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.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for skin irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.

 

For skin irritation, the substance demonstrated a negative potential for skin irritation response within an available skin irritation in vitro assay (OECD TG 439, 2018) with a relative mean MTT viability of 87.6%. In an available in vivo assay (OECD TG 404, 2018): mean scores for following grading at 24, 48 and 72h were 0.0, 2.0 and 1.0 in erythema and eschar and 0.0, 1.67 and 1.33 in edema scoring criteria. All effects had ceased in 2 of 3 females at day 14. All rabbits had score = 0 at the end of the observation. Slight desquamation was observed in 1 of 3 females. Applicant assessment indicates: based on the presented mean scores and reversibility of effects the substance is considered to meet the criteria for GHS Skin Irritation: category 3, causes mild skin irritation. This criterion is not implemented within the Regulation (EC) 1272/2008 criteria. The weight of evidence indicates that the substance has the potential to cause transient mild irritating effects but which are insufficient for classification.

 

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification based on one available in vitro assay (BCOP, OECD TG 437, 2018) and one in vivo assay (OECD TG 405, 2018).

 

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017)