Reaction mass of [(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde and [(3,7-dimethyloctyl)oxy]acetaldehyde

Endpoint summary

Description of key information

Skin irritation (OECD TG 439): Irritating

Skin corrosion (OECD TG 431): Not corrosive

Eye irritation (OECD TG 438): Irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2018 - 23 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number(s): 27958

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes exposure and 60 minute exposure: 37 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. After 1 hour of treatment the test item could not be removed completely.
- Observable damage in the tissue due to washing: no

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
The test substance was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Microplate reader: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 exposure times

EVALUATION
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls
(ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

PREDICTION MODEL / DECISION CRITERIA: see Table 1

ACCEPTABILITY CRITERIA:
1. The absolute mean OD570 of the two tissues of the negative control should reasonably be >= 0.8 and <= 2.8 and be within the laboratory historical control data range.
2. The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
3. In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be <= 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure / mean of 2 replicates

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

7.5

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure / mean of 2 replicates

Value:

89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

6.0

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.723 and 1.862)
- Acceptance criteria met for positive control: yes, the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 17%

Mean Absorption in the in vitro Skin Corrosion Test:

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.813	1.633	1.723	±	0.127	1.778	1.945	1.862	±	0.118
Muguet Aldehyde	1.722	1.641	1.682	±	0.058	1.510	1.815	1.662	±	0.216
Positive control	0.139	0.119	0.129	±	0.014	0.118	0.104	0.111	±	0.010

SD = Standard deviation

Duplicate exposures are indicated by A and B.

Mean Tissue Viability in the in vitro Skin Corrosion Test:

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Muguet Aldehyde	98	89
Positive control	7.5	6.0

Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	9.9	8.6
Muguet Aldehyde	4.8	17
Positive control	14	12

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Interpretation of results:

other: Not corrosive to the skin

Remarks:

In accordance with Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The results of an in vitro skin corrosion test showed that the substance was not corrosive to the skin.

Executive summary:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (Milli-Q water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to the substance were 98% and 89% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2018 - 16 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 18-EKIN-012 and 18-EKIN-015); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2.5 hours at 37°C.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.6 - 37.4 °C
- Humidity (%): 58 - 85

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed once with phosphate buffered saline
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20142750). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Muguet Aldehyde did not interfere with the MTT endpoint.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
- Cell viability: The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw): ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u +MTT).
The % viability for each sample and the positive control is calculated as follows: %Viability = (ODc/mean ODlt_u+MTT) * 100
- Skin irritation is expressed as the remaining cell viability after exposure to the test item.

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM:
25 μL directly on top of the tissue

NEGATIVE CONTOL:
- Amount applied: 25 μL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 μL
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3 for the test item, the negative and the positive control, each.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1

Value:

59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

13

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 2

Value:

39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

9

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Average experiment 1 and experiment 2

Value:

49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

11

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range and the SD of the % viability was ≤18%.
- Acceptance criteria met for positive control:
yes, the mean relative tissue viability of the positive control was <50% and the SD of the % viability was <18%.
- Acceptance criteria met for variability between replicate measurements:
yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <17%

- Based on all results together the substance result in a cell viability of 48.67%, which is just below the cut off value of 50% cell viability and therefore the substance is considered a skin irritant in absence of a third test.

Table Individual OD measurements (570 nm), experiment 1:

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	1.2637 1.2991	1.0774 1.0176	1.1547 1.0803
Test item OD570measurement 1 OD570measurement 2	0.4687 0.4651	0.8666 0.7975	0.7745 0.7828
Positive control OD570measurement 1 OD570measurement 2	0.1792 0.1730	0.3276 0.2761	0.0725 0.0710

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Table Individual OD measurements (570 nm), experiment 2:

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	0.9385 0.9203	0.9672 0.9008	0.9322 0.8985
Test item OD570measurement 1 OD570measurement 2	0.5695 0.5447	0.2920 0.2807	0.3256 0.3151
Positive control OD570measurement 1 OD570measurement 2	0.1283 0.1234	0.1036 0.1011	0.1488 0.1363

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Interpretation of results:

other: Skin irritant.

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

An in vitro skin irritation test with the substance was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is irritating in the in vitro skin irritation test.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. All validity criteria were met and the study was considered to be valid.

The positive control had a mean cell viability of 13% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 18%, indicating that the test system functioned properly. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 59%. Since the individual values were both above and below 50% (38, 71 and 67% respectively) the test was inconclusive and a repeat experiment was performed.

In the second test,the positive control had a mean cell viability of 9.0% after 15 ± 0.5 minutes exposure. The relative mean tissue viability obtained after 15±0.5 minutes treatment with the substance compared to the negative control tissues was 39%. The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly. The individual values were like in the first experiment both above and below 50% (58, 27 and 31% respectively).

Based on all results together the substance result in a cell viability of 48.67%, which is just below the cut off value of 50% cell viability and therefore the substance is considered a skin irritant in absence of a third test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jan 2018 to 05 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted on 9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Species:

other: Eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL neat substance

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Test group and positive control: triplicates
Negative control: Singlo

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA
According to OECD 438 guideline

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Other effects / acceptance of results:

- Slit-lamp examination: The test substance caused slight corneal swelling (mean score 11%), slight corneal opacity (mean score 1.0) and slight fluorescein retention (mean score 1.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
- Microscopic examination: Microscopic examination of the corneas treated with test substance generally revealed very slight erosion of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% generally revealed severe erosion of the epithelium and endothelial necrosis.

Interpretation of results:

other: Irritating to the eyes (category 2)

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is considered to be an eye irritant (category 2).

Executive summary:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL neat test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5% Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused slight corneal swelling (mean score 11%), slight corneal opacity (mean score 1.0) and slight fluorescein retention (mean score 1.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with test substance generally revealed very slight erosion of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% generally revealed severe erosion of the epithelium and endothelial necrosis. Based on these results, the substance has to be classified as irritating to the eyes (category 2).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. All validity criteria were met and the study was considered to be valid.

The positive control had a mean cell viability of 13% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 18%, indicating that the test system functioned properly. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 59%. Since the individual values were both above and below 50% (38, 71 and 67% respectively) the test was inconclusive and a repeat experiment was performed.

In the second test,the positive control had a mean cell viability of 9.0% after 15 ± 0.5 minutes exposure. The relative mean tissue viability obtained after 15±0.5 minutes treatment with the substance compared to the negative control tissues was 39%. The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly. The individual values were like in the first experiment both above and below 50% (58, 27 and 31% respectively).

Based on all results together the substance result in a cell viability of 48.67%, which is just below the cut off value of 50% cell viability and therefore the substance is considered a skin irritant in absence of a third test.

Skin corrosion:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (Milli-Q water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to the substance were 98% and 89% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.

Eye irritation:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL neat test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5% Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused slight corneal swelling (mean score 11%), slight corneal opacity (mean score 1.0) and slight fluorescein retention (mean score 1.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with test substance generally revealed very slight erosion of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% generally revealed severe erosion of the epithelium and endothelial necrosis. Based on these results, the substance has to be classified as irritating to the eyes (category 2).

Justification for classification or non-classification

Based on the results of an in vitro eye irritation study, the substance is classified as eye irritation category 2 and shall be labelled with H319: Causes serious eye irritation according to Regulation (EC) No. 1272/2008 and its amendments. The results of an in vitro skin irritation study also classified the substance as skin irritation category 2 and shall be labelled with H315: Causes skin irritation according to Regulation (EC) No. 1272/2008 and its amendments. The substance is not corrosive based on the results of an in vitro skin corrosion study.