Aluminium tribenzoate

Endpoint summary

Description of key information

An in vitro skin corrosion and irritation test were conducted with aluminium tribenzoate according to OECD 431 and 439 guidelines and GLP principles (Klimisch 1 studies). It is concluded that the test substance is not corrosive and not irritating to skin.

A Bovine Corneal Opacity and Permeability test (BCOP) is available performed according to OECD guideline 437 and GLP principles (Klimisch 1 study), which demonstrates that aluminium tribenzoate is not an eye irritant. 

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:

09 January 2017 - 19 January 2017

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:

other: EU method B.40 BIS (In Vitro Skin corrosion: Human Skin Model Test), adopted 31 May 2008

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Specific details on test material used for the study:

Purity correction factor: 1.164

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 24956 kit Q and R
- Surface: 0.6 cm^2

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C (actual range: 35.9 - 37.0°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

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

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration, one negative control, one positive contol.

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean viability of two tissues and one of the two tissues is ≤ 15%.

DECISION CRITERIA: see table 1

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent control for MTT reduction by test item

Amount/concentration applied:

26.56 - 45.10 mg of test item on top of skin moistened with 25 μl Milli-Q water

Duration of treatment / exposure:

3-minute and 1-hour

Duration of post-treatment incubation (if applicable):

none

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(100%)

Positive controls validity:

valid

Remarks:

(14%)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

111

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(100%)

Positive controls validity:

valid

Remarks:

(9%)

Other effects / acceptance of results:

- Direct-MTT reduction: No
- Colour interference with MTT: No

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: No, the Coefficient of Variation between tissue replicates of the negative control substance was 31% at the 1-hour treatment time which is not within the acceptability criteria of the assay (≤30%).

- Because the acceptability criterium for variability between replicate measurements was not met and the absolute OD570 (optical density at 570 nm) of the negative control tissues was not within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) for one of the skin tissues of the 3-minutes treatment time (0.754) and the absolute mean OD570 was not within the laboratory historical control data range both at the 3-minutes and 1-hour treatment time, a second set of negative controls was used. These negative controls were available from studies performed at the same time which fulfilled all acceptability criteria and could therefore be used for this study. The mean relative tissue viability following the 1-hour exposure to the positive control was 13% (first negative control) or 9% (second negative control). In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 25% with the second negative control, indicating that the test system functioned properly.

- Final results for tissue viability were based on the second set of negative controls.

- Table 1 shows the results of individual OD measurements at 570 nm.

Table 1 Mean absorption in the in vitro skin corrosion test with Aluminium tribenzoate

	3-minute application			1-hour application
	A (OD570)	B (OD570)	Mean (OD570­)±SD	A (OD570)	B (OD570)	Mean (OD570­)±SD
Negative control	0.754	0.891	0.822± 0.097	1.224	0.842	1.033± 0.270
Negative control (2)	1.707	1.380	1.543± 0.231	1.546	1.462	1.504± 0.060
Aluminium tribenzoate	1.720	1.291	1.505± 0.304	1.636	1.688	1.622± 0.037
Posutive control	0.271	0.172	0.222± 0.070	0.138	0.123	0.130± 0.011

SD = standard deviation

Duplicate exposures are indicated by A and B

In this table the values are corrected for background absorption (0.0418). Isopropanol was used to measure the background absorption.

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro skin corrosion test was conducted with Aluminium tribenzoate according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test under conditions described in this report.

Executive summary:

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model, Lot no.: 24956), the influence of aluminium tribenzoate on the viability of human skin was tested. At least twenty-five mg (actual dose: 26.56 - 45.10 mg) of Aluminium tribenzoate was applied directly to 0.6 cm² cultured skin which had been moistened with 25 μl of Milli-Q water. After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability after 3 minutes and 1 hour of exposure to the test item were 98% and 111%, respectively. The positive control had a mean cell viability of 9%. Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it can be concluded that Aluminium tribenzoate is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Feb 2017 - 06 Mar 2016

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 (incl. QA statement)

Remarks:

dd 03 November 2015

Specific details on test material used for the study:

Correction for purity: yes, a correction factor of 1.164 was applied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model
- Tissue batch number(s): 17-EKIN-009
- Surface: 0.38 cm^2

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 35.9 - 37.2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD: the test item was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model
- Dye used in the dye-binding assay: MTT

CELL VIABILITY MEASUREMENT
- Method: After incubation, cell culture inserts were dried and 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 dried and epidermis was separated from the collagen matrix. Both parts were placed in pre-labeled microtubes and extracted with 500 μl isopropanol. Tubes were stored refrigerated and protected from light for 71 hours. The amount of extracted formazan was determined spectrophotometrically.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 run, using 3 replicates

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered irritant in the skin irritation test 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.
A test item is considered non-irritant in the in vitro skin irritation test 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied: 11.1-11.5 mg directly on top of skin moistened with 5 μl Milli-Q water

NEGATIVE CONTROL
- Amount applied: 25 μl

POSITIVE CONTROL
- Amount applied: 25 μl
- The positive control was respread after 7 minutes contact time

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

114

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

12%

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 value of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: yes, the mean relative tissues viability of the positive control was < 50% (i.e. 12%)
- Acceptance criteria met for variability between replicate measurements: yes, SD between replicates was <18% (i.e. 7.6%)

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro skin irritation test with Aluminium tribenzoate was conducted according to OECD guideline 439 and GLP principles. It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model, Lot no.: 17-EKIN-009), the influence of Aluminium tribenzoate on the viability of human skin was tested. 11.1 to 11.5 mg Aluminium tribenzoate was applied directly to 0.38 cm² cultured skin (moistened with 5 μl of Milli-Q water). After 15 minutes, the substance was removed by washing and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 12% whereas the test substance showed cell viability of 114%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it is concluded that the test substance is not irritating in the in vitro skin irritation test.

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:

02 January 2017 - 03 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Specific details on test material used for the study:

Purity correction factor: 1.164
The test item was tested for solubility in physiological saline. Since no workable suspension of the test item in physiological saline could be obtained, it was used as delivered by the sponsor and added pure on top of the corneas.

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 317.8 to 354.9 mg (complete coverage of the cornea)

NEGATIVE CONTROL:
- Amount applied: 750 µL

POSITIVE CONTROL:
- Amount applied: 750 µL

Duration of treatment / exposure:

240 +/- 10 minutes

Duration of post- treatment incubation (in vitro):

90 +/- 5 minutes in sodium fluorescein

Number of animals or in vitro replicates:

3

Details on study design:

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) imidazole solution (positive control) were introduced onto the epithelium of the cornea. Aluminium tribenzoate was weighed and applied directly on the corneas in such a way that the cornea was completely covered (317.8 to 354.9 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red.
- POST-EXPOSURE INCUBATION: 90 ±5 minutes 32 ± 1 °C in sodium-fluorescein for permeability determinations.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

ACCEPTABILITY CRITERIA:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Run / experiment:

Single run/mean of 3 replicates

Value:

-1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS: 1.5 - 3.4

Positive controls validity:

valid

Remarks:

IVIS: 111.9 - 180.9

Other effects / acceptance of results:

The corneas treated with Aluminium tribenzoate showed opacity values of -2.2 and permeability values ranging from 0.023 to 0.067. IVIS scores were -1.4, -1.9 and -1.2 (n=3). The corneas were clear after the 240 minutes of treatment with Aluminium tribenzoate.

OTHER EFFECTS:
- The corneas treated with the positive control item were turbid after the 240 minutes of treatment.
- No pH effect of the test item was observed on the rinsing medium.
- The IVIS of one of the negative controls was >3. However, since the opacity, permeability and IVIS of this cornea were within the historical control data range and the test item gives a clear negative result this does not affect the study result.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (e.g. 1.5 - 3.4).
- Acceptance criteria met for positive control: yes, results were within historical range (e.g. 112 - 181).

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, it is concluded that aluminium tribenzoate is not classified under GHS (IVIS ranging from -1.9 to -1.2 after 240 minutes of treatment).

Executive summary:

The eye hazard potential of aluminium tribenzoate was assessed in a Bovine Corneal Opacity and Permeability test (BCOP test). Three corneas from young cattle were exposed to 317.8 - 354.9 mg (complete coverage) of the test item, next to a negative control group of physiological saline (n=3) and a positive control of 20% (w/v) imidazole (n=3). Duration of treatment was approximately 240 minutes. Results of the negative and positive controls were within the historical data. Therefore, it was shown that the negative control did not induce irritancy on the corneas, the test conditions were adequate and the test systems functioned properly. Aluminium tribenzoate did not induce ocular irritation, resulting in a mean in vitro irritancy score of -1.5 after 240 minutes of treatment. Based on this result (IVIS < 3), no classification for eye irritation or serious eye damage is required for aluminium tribenzoate according to GHS criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model, Lot no.: 24956), the influence of aluminium tribenzoate on the viability of human skin was tested. At least twenty-five mg (actual dose: 26.56 - 45.10 mg) of Aluminium tribenzoate was applied directly to 0.6 cm2 cultured skin which had been moistened with 25 μl of Milli-Q water. After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability after 3 minutes and 1 hour of exposure to the test item were 98% and 111%, respectively. The positive control had a mean cell viability of 9%. Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it can be concluded that Aluminium tribenzoate is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report. In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model, Lot no.: 17-EKIN-009), the influence of Aluminium tribenzoate on the viability of human skin was tested. 11.1 to 11.5 mg Aluminium tribenzoate was applied directly to 0.38 cm2 cultured skin (moistened with 5 μl of Milli-Q water). After 15 minutes, the substance was removed by washing and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 12% whereas the test substance showed cell viability of 114%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it is concluded that the test substance is not irritating in the in vitro skin irritation test.

The eye hazard potential of aluminium tribenzoate was assessed in a Bovine Corneal Opacity and Permeability test (BCOP test). Three corneas from young cattle were exposed to 317.8 - 354.9 mg (complete coverage) of the test item, next to a negative control group of physiological saline (n=3) and a positive control of 20% (w/v) imidazole (n=3). Duration of treatment was approximately 240 minutes. Results of the negative and positive controls were within the historical data. Therefore, it was shown that the negative control did not induce irritancy on the corneas, the test conditions were adequate and the test systems functioned properly. Aluminium tribenzoate did not induce ocular irritation, resulting in a mean in vitro irritancy score of -1.5 after 240 minutes of treatment.

Justification for classification or non-classification

Based on the available data, aluminium tribenzoate is not classified for skin or eye corrosive or irritant properties according to CLP Regulation (EC) No. 1272/2008.