Juniper, Juniperus mexicana, ext., isomerized, acetylated

Endpoint summary

Description of key information

Skin corrosion, in vitro: non-corrosive (3 -minutes viability 99% ; 1 -hour viability 90%), EPIDERM, OECD TG 431, 2015

Skin irritation, in vitro: irritating (15 minutes exposure/42hour incubation viability = 63%), EPISKIN, OECD TG 439, 2015

Eye irritation, in vitro: non-eye corrosive/serious irritating, mean IVIS = 1.1 and mean permeability = 0.010, OECD TG 437, 2015

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:

12-01-2015 to 21-05-2015

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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 50%) which does not affect the reliability of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 50%) which does not affect the reliability of the study

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

In accordance with the OECD TG 431 guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): Lot no.: 21267 kit Q
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 12-01-2015 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 12-01-2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C, 5 ± 0.5% CO2 (actual: 36.5 – 37.4 °C)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.0 °C, 5 ± 0.5% CO2; for 3 hours. For further information see below.

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. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed. For the cell viability measurements: The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. Prior to spectrophotometric analysis for extracted formazan.
- 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: 5.0 mg/mL MTT concentrated diluted (1:5) in MTT diluent solution prepared in assay medium
- Incubation time: For the cell viability measurements: The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. Prior to spectrophotometric analysis for extracted formazan.
- Spectrophotometer: M200 Pro Plate Reader
- Wavelength: 540 nm
- 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 positive control had a mean relative tissue viability of 10% after the 1-hour exposure. The absolute mean OD540 of the negative control tissues was
within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 11%, indicating that the test system functioned properly.
- 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: Two (2), duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the previously conducted pretest for direct MTT reduction and colour interference (conducted in separated study cited in full study report): the solution containing the test item was colourless and/or solutions did not turn blue / purple and a blue / purple precipitate was not observed 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) for exposure time 3 minutes and exposure time 1-hour

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if : the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if: the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Cut off points in accordance with OECD TG 431 and the GHS and CLP Classification systems.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: Not applicable.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test substance at exposure times 3-minutes and/or 1-hour with 3-hours incubation. Where necessary, direct MTT reduction, colour interference was completed.

OTHER:
EpiDerm Skin Model (EPI-200, Lot no.: 21267 kit Q). The model 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) were cultured on polycarbonate membranes of 10 mm cell culture inserts. All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Preincubation:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.

Application of test item and rinsing:
The plates were incubated for approximately 3 hours at 37 ± 1.0°C. The medium was replaced with fresh DMEM just before the test item was applied. Two tissues were used for a 3-minute exposure and two for a 1-hour exposure. Test item: 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues. Negative Control: similarly treated with 50 µl Milli-Q water only. Positive control: 50 µL 8N KOH (potassium hydroxide 8 normal solution). After the exposure periods (3 minutes and 1 hour, respectively), the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed. All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): Not applicable (Milli-Q water)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N KOH(aq) (pre-diluted)

Duration of treatment / exposure:

Observations are made 3-minutes and 1-hour post-test item application

Duration of post-treatment incubation (if applicable):

The DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature.

Number of replicates:

Duplicate; used for a 3-minute exposure to the test substance and two for a 1-hour exposure.

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minute exposure

Run / experiment:

mean (n=2)

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 3 minute exposure. Remarks: n=2 ; SD = 7.0 % ; Score in terms of percentage of negative control.

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour exposure

Run / experiment:

mean (n=2)

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 60 minute exposure. Remarks: n=2 ; SD = 2.2 % ; Score in terms of percentage of negative control.

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

Dose Group	Replicate	3 minute OD540 nm corrected (for blank)	Mean OD540 nm	Standard Deviation	3 minute Mean viability (% of control)	CoV (%)	1 hour OD540 nm corrected (for blank)	Mean OD540 nm	Standard Deviation	1 hour Mean viability (% of control)	CoV (%)
Blank (isopropanol)		0.043
Negative Control	A	1.719	1.676	0.060	100	3.58	1.718	1.680	0.054	100	3.21
	B	1.634					1.641
Positive Control	A	0.133	0.130	0.005	8	3.85	0.184	0.166	0.026	10	15.6
	B	0.126					0.148
Test Item	A	1.737	1.655	0.117	99	7.07	1.533	1.508	0.034	90	2.25
	B	1.573					1.484

 

The positive control had a mean relative tissue viability of 10% after the 1-hour exposure. The absolute mean OD540 of the negative control tissues was within the acceptance limits of OECD TG 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 11%, indicating that the test system functioned properly.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 study, the test item is not considered to be corrosive to the skin.

Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of four tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μL of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 10% after the 1-hour exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the acceptance limits of OECD TG 431 (lower acceptance limit≥0.8 and upper acceptance limit≤2.8) and the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 20% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 11%, indicating that the test system functioned properly. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was≤11%, indicating that the test system functioned properly. All assay acceptance criteria were met. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99% and 90%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not considered to be corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-03-2015 to 09-03-2015

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:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 79%) which does not affect the reliability of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 79%) which does not affect the reliability of the study

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

In accordance with the OECD TG 439 guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model
- Tissue batch number(s): Lot no.: 15-EKIN-009
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 03-03-2015 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 03-03-2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C, 5 ± 0.5% CO2 (actual: 36.3 – 36.6°C)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.0 °C, 5 ± 0.5% CO2; for 42 hours. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 69 to 72 hours. The amount of extracted formazan was determined spectrophotometrically.
- 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: 3.0 mg/mL MTT concentrated diluted (x10) to 0.3 mg/mL MTT solution prepared in assay medium
- Incubation time: For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 69 to 72 hours. The amount of extracted formazan was determined spectrophotometrically.
- Spectrophotometer: M200 Pro Plate Reader
- Wavelength: 570 nm
- 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 7% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 6.9%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.219 and the standard deviation of viability was 4.1%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 27.8%. 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. Applicant assessment indicates that the concurrent positive control was towards the lower range of the HCD (0.020 – 0.408, mean: 0.14, SD: 0.11 and n=94), whereas the negative controls were toward the upper range of the HCD (0.576 – 1.352, mean: 1.02, SD: 0.15 and n=94). This may be a contributing cause of the > 18% SD observed in the test item replicates. Additionally, the SD from test item treated individual tissue viabilities is 14.4% and no individual tissues indicated a positive (< 50%) result relative to the negative control. Whereas the positive control indicated a clear skin irritation response = 7%.

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 15-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 15-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 15-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference modifications were completed.

OTHER:
EpiSkin RHE Small Model (Lot no.: 15-EKIN-009). The test consists of topical application of the test item -one on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is 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 the treatment. The EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37°C. Maintenance medium and Assay medium were supplied by a recognised supplier (documented in the full study report).

Application of test item and rinsing:
Test item: Tissues were treated with 10 µL test item for 15 minutes exposure period. Negative control: 10 µL PBS (negative control) was similarly applied. Positive control: 10 µLl SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 79 – 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 – 36.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): Not applicable. (Phosphate buffered saline, pre-diluted)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% SDS (pre-diluted)

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature, after which the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

Triplicate; treatment and concurrent negative control and positive control groups

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (n=3)

Value:

63

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute exposure. Remarks: n=3; SD = 27.8% however test item treated individual tissue viabilities SD = 14.4% ; 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. Applicant assessment indicates that the concurrent positive control was towards the lower range of the HCD (0.020 – 0.408, mean: 0.14, SD: 0.11 and n=94), whereas the negative controls were toward the upper range of the HCD (0.576 – 1.352, mean: 1.02, SD: 0.15 and n=94). This may be a contributing cause of the > 18% SD observed in the test item replicates. Additionally, the SD from test item treated individual tissue viabilities is 14.4% and no individual tissues indicated a positive (< 50%) result relative to the negative control. Whereas the positive control indicated a clear skin irritation response = 7%
- 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. Historic Control Data (n=94): the mean OD of the positive control was 0.14 ; range 0.020 – 0.408. In this same period the mean OD of the negative control was 1.02 ; range 0.576 – 1.352.

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

	OD570			Mean	SD	Mean tissue viability (% of control)
	A	B	C
Negative control	1.227	1.249	1.180	1.219	±0.035	100
Test Material	1.015	0.669	0.623	0.769	±0.214	63
Positive control	0.094	0.084	0.083	0.087	±0.006	7

OD = optical density SD = Standard deviation Triplicate exposures are indicated by A, B and C.

Values are corrected for background adsoption (0.042). Isopropanol was used to measure background adsorption.

Negative control: Phosphate buffered saline (PBS)

Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

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 considered to be not irritating to the skin.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of three tissues per test item together with negative and positive controls. 10 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μL PBS (negative control) and 3 tissues with 10 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability 7% after 15 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 relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 63%. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is not considered to be irritant. Under the conditions of this study the test item is not irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-01-2015 to 21-05-2015

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

Qualifier:

according to guideline

Guideline:

other: The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

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. Post-excision, the isolated eyes were stored in physiological saline in the cooled slaughter-house until transportation to the laboratory using a suitable container. The corneae were isolated on the same day after delivery of the eyes.
- 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: None reported.

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 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

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. The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

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: physiological saline solution

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: 10% (w/v) Benzalkonium Chloride solution prepared in physiological saline.

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

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

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 (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 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:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

IVIS scores ranged from 1.0 to 1.3 after 10 minues treament

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None. No pH effect of the test item was observed on the rinsing medium.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. 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.
- Acceptance criteria met for positive control: Yes. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 128.2 and was within the historical positive control data range.
- Range of historical values if different from the ones specified in the test guideline: The mean in vitro irritancy score of the negative control were less than the upper limits of the laboratory historical range and for the positive control (10% (w/v) Benzalkonium Chloride) at 128.2 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

Table 1 : Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity #1	Mean Permeability #1	Mean In vitro Irritation Score #1 , 2
Negative Control	0	0.000	0.0
Positive Control (Benzalkonium chloride)	81.7	3.102	128.2
Test item	1.0	0.010	1.1

#1 Calculated using the negative control mean opacity and mean permeability values

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2 : Opacity score

Eye	Opacity before treatment	Opacity after treatment	Final opacity #1	Negative control corrected final opacity #2	Mean Opacity
Negative Control
1	0	0	0	0	0
2	0	0	0	0
3	0	0	0	0
Positive Control
4	0	83	84	84	81.7
5	0	83	84	84
6	0	76	77	77
Test item
13	0	1	1	1.0	1.0
14	-1	0	1	1.0
15	-1	0	1	1.0

#1 Final Opacity = Opacity after treatment – Opacity before treatment

#2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

 

Table 3 Permeability score individual values (uncorrected)

Eye	Dilution Factor	OD490 1	OD490 2	OD490 3	Average OD490	Final OD	Mean Final Negative Control
	Negative Control
1	1	0.010	0.011	0.011	0.011	0.011	0.008
2	1	0.004	0.004	0.004	0.004	0.004
3	1	0.009	0.007	0.008	0.008	0.008
	Positive Control
4	6	0.314	0.311	0.312	0.312	1.874
5	6	0.482	0.479	0.473	0.478	2.868
6	6	0.794	0.781	0.775	0.783	4.700
	Test item
13	1	0.022	0.033	0.024	0.026	0.026
14	1	0.009	0.011	0.012	0.011	0.011
15	1	0.013	0.013	0.018	0.015	0.015

 

Table 4 : In vitro irritancy score

Eye	Negative control corrected final opacity	Negative control corrected Final OD490	In vitro Irritancy Score #1
Negative Control
1	0.0	0.003	0.0
2	0.0	-0.004	-0.1
3	0.0	0.000	0.0
Positive Control
4	84.0	1.829	111.4
5	84.0	2.823	126.3
6	77.0	4.655	146.8
Test item
13	1.0	0.019	1.3
14	1.0	0.003	1.0
15	1.0	0.007	1.1

#1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value)

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 item in isolated bovine corneas. The purpose of this test was to evaluate the test item for its potential to induce serious eye damage/ocular irritation. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. A total of three corneas per treatment group were used. A volume of 750 microlitres of the item was placed the cornea. The negative control group received saline and the positive control group received 10% benzalkonium chloride. For each group the corneas were incubated for 10 minutes at 32 degrees C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120±10 minutes at 32±1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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 (10% (w/v) Benzalkonium Chloride) was 128.2 (GHS Category 1 : eye damage prediction) and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment. Based on these results the item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion :

Key study : in vitro, OECD TG 431, 2015 : The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of four tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μL of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 10% after the 1-hour exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the acceptance limits of OECD TG 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 20% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 11%, indicating that the test system functioned properly. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤11%, indicating that the test system functioned properly. All assay acceptance criteria were met. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99% and 90%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not considered to be corrosive to the skin.

 

Skin Irritation :

Key study : in vitro, OECD TG 439, 2015 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of three tissues per test item together with negative and positive controls. 10 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μL PBS (negative control) and 3 tissues with 10 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability 7% after 15 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 relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 63%. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is not considered to be irritant. Under the conditions of this study the test item is not irritant to the skin.

 

Eye irritation/corrosion :

Key study : in vitro, OECD TG 437 2015 : 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 item in isolated bovine corneas. The purpose of this test was to evaluate the test item for its potential to induce serious eye damage/ocular irritation. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. A total of three corneas per treatment group were used. A volume of 750 microlitres of the item was placed the cornea. The negative control group received saline and the positive control group received 10% benzalkonium chloride. For each group the corneas were incubated for 10 minutes at 32 degrees C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120±10 minutes at 32±1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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 (10% (w/v) Benzalkonium Chloride) was 128.2 (GHS Category 1 : eye damage prediction) and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment. Based on these results the item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

Respiratory Irritation:

No data available.

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