bis[C11-14-(branched and linear)-alkyl]aminium nonadecaoxohexatungstate

Endpoint summary

Description of key information

Skin irritation:
Bis[C11-14-(branched and linear)-alkyl]aminium nonadecaoxohexatungstate (EC 700-718-0), based upon results from the surrogate substance, is not irritating to skin.
Eye irritation / corrosion:
Bis[C11-14-(branched and linear)-alkyl]aminium nonadecaoxohexatungstate (EC 700-718-0), based upon results from the surrogate substance, is not irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22.12.2011 - 06.04.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to OECD Guideline under GLP conditions.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 22 July 2010).

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

other: adult human-derived epidermal keratinocytes.

Details on test animals or test system and environmental conditions:

Not applicable

Controls:

other: Not applicable

Amount / concentration applied:

Not applicable

Duration of treatment / exposure:

Not applicable

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

Test substance preparation
The test substance has a sticky consistence and was applied using a cotton swab directly on top of the skin tissue. The test substance was spread to match the size of the tissue.

Reference substances

Negative control:
Phosphate buffered saline (PBS, Invitrogen Corporation,, The Netherlands).
 
Positive control:
5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands) [CAS Number 151-21-3] in PBS.

Test system

EPISKIN Small Model (TM) (EPISKIN-SM(TM), 0.38 cm2, Batch no.: 12-Ekin-011).
This 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.

Source: SkinEthic Laboratories, Lyon, France.

Preparation and preincubation

Tissue
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37 degrees Centigrade. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Nice, France.

Killed tissue (EPISKIN-SM)TM), 0.38 cm2, Lot no.: 12 Ekin-007, received: 14 February, 2012)
Living epidermis was transferred to 12-well plates and incubated with 2 ml Milli-Q for 48 +/- 1 hours. After incubation, killed epidermis was stored at <= 15 degrees Centigrade. Killed tissues were thawed by placing them for 1 hour at room temperature in 12-well plateson 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.

MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10 x) in Assay medium (final concentration of 0.3 mg/ml).

Environmental conditions
All incubations, with the exception of the test substance 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 75 - 97%), containing 5.0 +/- 0.5% CO2 in air in the dark at 37.0 ± 1.0 degrees Centigrade (actual range 35.2 - 37.6 degrees Centigrade). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.2 - 36.0 degrees Centigrade) and humidity (with a maximum of 5%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Study design

Test for reduction of MTT by the test substance
The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 21 mg of the test substance was added to a 12 well plate filled with 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37 degrees Centigrade. A negative control, sterile Milli-Q water was tested concurrently.

In case the test substance reacts with the MTT medium in addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues must be used for the cytotoxicity evaluation with MTT.

Application/Treatment of the test substance
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The undiluted test substance was added (using a cotton swab) into 12-well plates on top of the skin tissues. Due to the sticky consistence of the test substance, dead tissue was separated from the collagen matrix and the skin was damaged. Three tissues were treated with 25 microL PBS (negative control) and 3 tissues with 25 microL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. 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 44 hours at 37 degrees Centigrade.

Cell viability measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37 degrees Centigrade. 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 prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Irritation / corrosion parameter:

other: other: tissue viability

Value:

125

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 100.0. Remarks: scores as percentage relative to negative control (100%). (migrated information)

Irritant / corrosive response data:

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that the test substance did interact with MTT.

In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test substance was 5% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 125%. Since the mean relative tissue viability for the test substance was above 50% the test substance is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 5%. The absolute mean OD570 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 less than 8%, indicating that the test system functioned properly.

Mean tissue viability with test substance

	Mean tissue viability (percentage of control)
Negative control	100
Test substance	125
Positive control	5

Individual OD measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	0.950 0.989	0.983 1.010	1.012 1.043
Test substance on viable tissue OD570measurement 1 OD570measurement 2	0.987 1.539	1.116 1.346	1.264 1.483
Test substance on killed tissue OD570measurement 1 OD570measurement 2	0.104 0.204	0.130 0.127	0.116 0.156
Non treated killed tissue OD570measurement 1 OD570measurement 2	0.073 0.104	0.072 0.113	0.075 0.085
Positive control OD570measurement 1 OD570measurement 2	0.087 0.093	0.081 0.107	0.084 0.099

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an OECD 439 study (In Vitro Skin Irritation: Reconstituted Human Epidermis Test Method), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is a non-irritant (Notox B.V., 2012).

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22.12.2011 - 2.3.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to OECD Guideline under GLP conditions.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

other: Bovine eyes from young cattle

Details on test animals or tissues and environmental conditions:

Not applicable

Amount / concentration applied:

Not applicable

Duration of treatment / exposure:

Not applicable

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

Test substance

Test substance preparation
The test substance was tested undiluted.

Reference substances

Negative control:
A negative control, physiological saline (Merck, Darmstadt, Germany) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

Positive control:
10% (w/v) Benzalkonium Chloride (sigma-Aldrich Chemie GmbH, Germany) [CAS Number 63449-41-2] solution prepared in physiological saline.

Test system

Test system
Bovine eyes were used as soon as possible after slaughter on the same day.

Source
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport
Eyes were collected and transported in physiological saline in a suitable container.

Study design

Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation were discarded.

The isolated corneas were stored at 32 +/- 1 degrees Centigrade in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, 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 degrees Centigrade. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 degrees Centigrade.

Cornea selection and Opacity reading
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 (OP-KIT, MC2, Clermont, France). 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.

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 microL of the negative and positive control substances were introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. The test substance was applied by using a plastic Pasteur Pipette till the cornea was completely covered. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1 degrees Centigrade. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) 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. Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1 degrees Centigrade. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck, Darmstadt, Germany) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1 degrees Centigrade.

Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 microL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Irritation parameter:

other: opacity

Basis:

mean

Score:

2

Max. score:

7

Irritation parameter:

other: permeability

Basis:

mean

Score:

0.037

Max. score:

0.103

Irritation parameter:

other: In vitro irritation

Basis:

mean

Score:

2.6

Max. score:

4.4

Irritant / corrosive response data:

The corneas treated with the test substance showed opacity values ranging from 0.008 to 0.094. The corneas were clear after the 10 minutes of treatment with the test substance. Hence, the in vitro irritancy scores ranged from 0.1 to 4.4 after 10 minutes of treatment with the test substance.

The mean in vitro irritancy scores for the negative controls was 0. The individual positive control in vitro irritancy scores ranged from 113 to 132 for Benzalkonium Chloride. The corneas treated with the positive control substances were turbid after 10 minutes of treatment.

The negative control response 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 cornea. The mean in vitro irritancy score of the positive control (10% (w/v) enzalkonium Chloride) was 119 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.

Table 1           Summary of opacity, permeability and in vitro scores

 

Treatment	Mean Opacity^1	Mean Permeability^1	MeanIn vitro Irritation Score^1, 2
Negative control	0	0.000	0.0
Positive control (Benzalkonium Chloride)	67	3.486	119
Test substance	2	0.037	2.6

 

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

2           In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

 

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an OECD 437 study (Bovine Corneal Opacity and Permeability (BCOP) test), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is a non-irritant (Notox B.V., 2012).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation /corrosion

In an OECD 439 study (In Vitro Skin Irritation: Reconstituted Human Epidermis Test Method), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is a non-irritant (Notox B.V., 2012).

Eye irritation / corrosion

In an OECD 437 study (Bovine Corneal Opacity and Permeability (BCOP) test), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is a non-irritant (Notox B.V., 2012).

Respiratory irritation:

No information is available on respiratory information.

Justification for selection of skin irritation / corrosion endpoint:
Accepted in vitro OECD 439 Guideline study, conducted to GLP.

Justification for selection of eye irritation endpoint:
Accepted in vitro OECD 437 Guideline study, conducted to GLP.

Justification for classification or non-classification

Skin irritation

In an in vitro study, mean tissue viability was found to be 125%.  Based on the very low scores the substance is considered not irritating to skin, therefore, according to Table 3.2.2.6 Decision logic for classification of substances of Regulation EC No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures the substance is not classified.

Eye irritation

In an in vitro study, a mean irritancy score of 2.6 after 10 minutes of treatment was achieved. Based on the low mean eye irritancy score the substance is considered not irritating to eyes, therefore, according to Table 3.3.2.6 Decision logic of Regulation EC No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures the substance is not classified.