Vinyltoluene

Endpoint summary

Description of key information

Skin corrosion: Not corrosive (OECD431/GLP)

Skin irritation: Irritating (OECD 439/GLP)

 

Serious eye damage/eye irritation: No prediction can be made (OECD 437/GLP)

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:

05 October 2020 - 16 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EC method B.40 bis: In vitro Skin Corrosion: Human Skin Model Test Council Regulation (EC) No. 2019/1390, adopted 31 July 2019.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Batch no.: 20200608
- Expiration: 07 June 2021
- Purity, including information on contaminants, isomers, etc.: 99.66% (3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at +2 °C to +8 °C in a tightly closed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none, the test item was used as supplied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s):Lot no. 30897
- Production date: October 14,2020
- Date of initiation of testing: October 13,2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37℃

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 18 times in approximately 100 mL D-PBS
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan infinite 200Pro10
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD[540-570nm] (1.0-3.0):1.906±0.071
- Barrier function: ET-50[4.77-8.72 hrs]:6.52 hrs
- Contamination: Sterile

NUMBER OF REPLICATE TISSUES:
Two tissues were used per treatment for test item, positive and negative controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE/COLOUR INTERFERENCE
1. Functional check for MTT interference was performed as follows: Fifty μL of the test item were added to 1 mL of the MTT medium (1 mg MTT6/mL) and incubated at 37 °C, 5 % CO2, and 95 % relative humidity for 60 minutes. Untreated MTT solution was used as control. The test item displayed a slight purple discolouration. Due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed. The frozen tissues were stored in the freezer (-20 ± 5 °C). The test item was applied to two freeze-killed tissues9. In addition, two freeze-killed tissues were treated with the negative control. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDermTM tissues.

2. Functional check for colour interference was performed as follows: Fifty μL of the test item were mixed with 300 μL sterile deionised water and incubated at 37 °C, 5 % CO2 and 95 % relative humidity for 60 minutes. In addition, 50 μL of the test item were added to 2 mL isopropanol and incubated at room temperature for three hours. No discolouration of the test item was observed in either case.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if [complete, e.g. 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%.]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Fifty µL


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Fifty µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Fifty µL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 mins

Value:

85.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 85.4 % ± 3.7

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction and Colour interference with MTT: No discolouration of the test item with water or isopropanol was noted.

As the test item displayed a slight purple discolouration due to interaction of the test item with the MTT measurement (reduction of MTT), an additional test with freeze-killed tissues had to be performed. The OD correction factor for the 3-minute exposure was 0.0034, while the OD correction factor for the 1-hour exposure was determined as 0.0295.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control and positive control:
The mean optical density (OD) of the negative control of 2 tissues was 1.7940 (3-minute exposure) or 1.7560 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.

The mean viability of cells treated with the positive reference item 8 N KOH was 6.7 % ± 0.1 % or 4.0 % ± 0.2 % (3-minute or 1-hour exposure, respectively) of the negative control and, hence well below the 15 % cut-off value at the 1-hour exposure.

- Acceptance criteria met for variability between replicate measurements:
The difference of viability between two tissue replicates (for tissues in the viability range between 20 – 100 % viability) was below the limit of acceptance of 30 %. Hence, all acceptance criteria were fulfilled

Historical data of negative and positive controls are presented in Appendix 3.

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro skin corrosion test using the reconstructed human epidermal model EpiDerm, the test item Vinyltoluene was non-corrosive.

Executive summary:

In an in vitro skin corrosion in the human epidermal model EpiDerm (OECD 431/GLP), reconstructed human epidermis tissue was exposed to 50 µL of Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) for 3 and 60 minutes. Sterile deionised water was used for the negative control and 8N KOH was used for the positive control. After removal of the test substance, the tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol overnight at room temperature with shaking. The OD540 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean absolute OD570 of the 2 negative control tissues was ≥ 0.8 and ≤ 2.8 [1.7940 (3 min) and 1.7560 (60 mins)]. The mean relative tissue viability (% negative control) of the positive control was < 15% [6.7 % ± 0.1 % or 4.0 % ± 0.2 % (3-minute or 60 mins exposure, respectively)]. The colour of the test substance did not interfere with the endpoint. As the test item displayed a slight purple discolouration due to interaction of the test item with the MTT measurement (reduction of MTT), an additional test with freeze-killed tissues had to be performed. The OD correction factor for the 3-minute exposure was 0.0034, while the OD correction factor for the 1-hour exposure was determined as 0.0295.

 

The average viability of tissues treated by the test item, 1 Vinyltoluene were higher than the threshold values (50 % and 15% respectively): 85.4 % ± 3.7 after a 3-minute exposure period and 63.5 % ± 2.7 after a 1-hour exposure. The test item should be regarded as non-corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2020 - 02 December 2020

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Batch no.: 20200608
- Expiration: 07 June 2021
- Purity, including information on contaminants, isomers, etc.: 99.66% (3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at +2 °C to +8 °C in a tightly closed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none, the test item was used as supplied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ EPI-200
- Tissue batch number(s): Lot no. 30891
- Production date: September 9,2020
- Date of initiation of testing: 09 September 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 60 minutes (The incubation conditions were in total 25 minutes at room temperature under a sterile hood and 35 minutes at 37 °C, 5 % CO2 and 95 % relative humidity).
- Temperature during post-treatment:37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Infinite 200 Pro, Magellan Version 7.2
- Wavelength: 540nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD[540-570nm](1.0-3.0): 1.9±0.165
- Barrier function: ET-50[4.77-8.72hrs]:5.45 hrs
- Contamination: Sterile

NUMBER OF REPLICATE TISSUES: 3 tissues for test item, negative and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE/COLOUR INTERFERENCE
1. The test item was evaluated for the potential to interfere with the MTT assay reagent (e.g. reduction). A concurrent negative control (sterile deionised water) was run in parallel. Thirty μL test item was added to 1 mL MTT solution5 and incubated at 37 °C, 5 % CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. No discolouration of the test item was noted by visual inspection.
2. Prior to the testing, the test item was evaluated for colour changes. Concurrent negative controls (sterile deionised water) were run in parallel. Thirty μL test item was mixed with 300 μL sterile deionised water3 and incubated in the dark at 37 °C, 5 % CO2 and 95 % relative humidity for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolouration of the test item was noted by visual inspection. In addition, 30 μL test item were mixed with 2 mL isopropanol4 and incubated at room temperature for three hours. No discolouration of the test item/isopropanol suspension was noted by visual inspection. Hence, the criteria of the test on colour interference were met.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritation to skin if [mean tissue viability ≤ 50 % Irritant (I), (H314 or H315 or GHS Category 1 or 212) ;
mean tissue viability > 50 % non-irritant (NI)..]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42-hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

12.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 12.3% ± 4.6 %

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction and Colour interference with MTT:
The mixture of test item with water or isopropanol did not show any discolouration nor did the test item itself directly interact with MTT. Therefore, no quantitative correction of assay results was necessary.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control and positive control:
The mean optical density (OD) of 3 negative control tissues was 1.2530 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.

The viability of cells treated with the positive reference item, 5 % SDS, was 6.3 % ± 0.1 % of the negative control and fulfilled the acceptance criterion of ≤ 20 %.

- Acceptance criteria met for variability between replicate measurements:
The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was below the limit of acceptance of 18 %.

Historical data of negative and positive controls are presented in Appendix 3.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In the in vitro skin irritation test using the reconstructed human epidermal model EpiDerm, the test item Vinyltoluene is considered a skin irritant.

Executive summary:

In an in vitro skin irritation assay in a human epidermal model EpiDerm (OECD 439/GLP), reconstructed human epidermis tissue was exposed to 30 µL of Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at 35 minutes at 37 °C, 5 % CO2 and 95 % relative humidity). DPBS was used for the negative control and 5% aqueous SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for 42 hours. After three hours incubation with MTT, samples were extracted with isopropyl alcohol for approximately two hours. The OD540 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.2530). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (6.3% ± 0.1 The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was below the limit of acceptance of 18 %. The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing.

 

The average viability of tissues treated by the test item, Vinyltoluene, was 12.3% ± 4.6 % of the negative control average value i.e. viability was < 50 %. Combined with the non-corrosion results of OECD431, the test item is classified as UN GHS category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2020 - 03 February 2021

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)

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 (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Batch no.: 20200608
- Expiration: 07 June 2021
- Purity, including information on contaminants, isomers, etc.: 99.66% (3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at +2 °C to +8 °C in a tightly closed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none, the test item was used as supplied.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from cattle were obtained from a slaughterhouse (Otto Vollertsen GmbH, 24986 Mittelangeln, Germany)
- Characteristics of donor animals (e.g. age, sex, weight): 6 to 12 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):To minimise deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks' Balanced Salt Solution 3 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL
- Selection and preparation of corneas: The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period had to be discarded.
- Quality check of the isolated corneas: Yes

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):750 µL (undiluted); closed-chamber method.


VEHICLE
none

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9 % NaCl solution

SOLVENT CONTROL USED (if applicable): none

POSITIVE CONTROL USED: 1 % NaOH solution in highly purified water

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least two times until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber.

- POST-EXPOSURE INCUBATION: The holder was incubated in a horizontal position at 32 ± 1 °C for 2 hrs.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with an opacitometer (BASF 2011-13; 67063 Ludwigshafen am Rhein, Germany) resulting in opacity values measured on a continuous scale.

- Corneal permeability: 1 mL sodium fluorescein solution (4 mg/mL in 0.9 % sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Infinite M200 Pro12). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 based upon a 96-well microtiter plate reader (Tecan Infinite M200 Pro).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: IVIS ≤ 3 : No Category; IVIS> 3 and ≤ 55 : No prediction can be made ; IVIS > 55: Category 1

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

1.992

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 1.992 ±0.222

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

7.927

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 7.927±2.006

Irritation parameter:

fluorescein leakage

Run / experiment:

1

Value:

0.396

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 0.396 ±0.134

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The corneas treated with the negative control item 0.9 % NaCl solution revealed a mean opacity value of 0.478 ± 1.679 and a mean permeability value of 0.009 ± 0.004. The calculated IVIS value of 0.613 ± 1.731 was well below the cut-off value of 3 (UN GHS no category). One value (2.231) of the triple determination of opacity values of the negative control was slightly higher than the upper limit of acceptance value of the historical background data (1.604). As this value is only slightly higher than the upper limit of acceptance of the historical background data and hence, is not an extreme outlier, this value is acceptable for inclusion into the historical background data. Additionally, the mean opacity value of the negative control is lower than the upper limit of acceptance of the historical control (Appendix 2).

- Acceptance criteria met for positive control: The corneas treated with the positive control item 1 % NaOH in highly purified water solution revealed a mean opacity value of 31.634 ± 10.847 and a mean permeability value of 3.024 ± 1.363 compared to the solvent control. The calculated IVIS value of 76.999 ± 10.459 was within two standard deviations of the current historical mean (Appendix 2) and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Interpretation of results:

study cannot be used for classification

Conclusions:

In the Bovine Corneal Opacity and Permeability (BCOP) assay, the IVIS value for Vinyltoluene is 7.927 ± 2.006 (> 3 and ≤ 55), therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.

Executive summary:

In the in vitro eye irritation Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), 3 isolated bovine corneas were exposed to Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) in liquid form for 10 minutes using the closed-chamber method. 0.9% sodium chloride was used for the negative control and 1% NaOH solution in highly purified water was used for the positive control. The corneas were rinsed and following a 2-hour post-exposure incubation at 32°C ± 1°C, the opacity and permeability of each cornea was recorded.

 

The positive and negative controls gave the appropriate response (IVIS = 76.999 ± 10.459 and 0.613 ± 1.731, respectively). The mean opacity value for the test substance was 1.992 ± 0.222. The mean permeability OD490 for the test substance was 0.396 ± 0.134. The IVIS for the test substance was 7.927 ± 2.006. The IVIS for Vinyltoluene is > 3 and simultaneously ≤ 55, therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion

There is one in vitro skin corrosion study available.

 

In an in vitro skin corrosion in the human epidermal model EpiDerm (OECD 431/GLP), reconstructed human epidermis tissue was exposed to 50 µLof Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) for 3 and 60 minutes. Sterile deionised water was used for the negative control and 8N KOH was used for the positive control. After removal of the test substance, the tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol overnight at room temperature with shaking. The OD540 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean absolute OD570 of the 2 negative control tissues was ≥ 0.8 and ≤ 2.8 [1.7940 (3 min) and 1.7560 (60 mins)]. The mean relative tissue viability (% negative control) of the positive control was < 15% [6.7 % ± 0.1 % or 4.0 % ± 0.2 % (3-minute or 60 mins exposure, respectively)]. The colour of the test substance did not interfere with the endpoint. As the test item displayed a slight purple discolouration due to interaction of the test item with the MTT measurement (reduction of MTT), an additional test with freeze-killed tissues had to be performed. The OD correction factor for the 3-minute exposure was 0.0034, while the OD correction factor for the 1-hour exposure was determined as 0.0295.

 

The average viability of tissues treated by the test item, 1 Vinyltoluene were higher than the threshold values (50 % and 15% respectively): 85.4 % ± 3.7 after a 3-minute exposure period and 63.5 % ± 2.7 after a 1-hour exposure. The test item should be regarded as non-corrosive.

 

Skin irritation

 

There is one in vitro skin irritation study available.

 

In an in vitro skin irritation assay in a human epidermal model EpiDerm (OECD 439/GLP), reconstructed human epidermis tissue was exposed to 30 µL of Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at 35 minutes at 37 °C, 5 % CO2 and 95 % relative humidity). DPBS was used for the negative control and 5% aqueous SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for 42 hours. After three hours incubation with MTT, samples were extracted with isopropyl alcohol for approximately two hours. The OD540 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.2530). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (6.3% ± 0.1 The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was below the limit of acceptance of 18 %. The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing.

 

The average viability of tissues treated by the test item, Vinyltoluene, was 12.3% ± 4.6 % of the negative control average value i.e. viability was < 50 %. Combined with the non-corrosion results of OECD431, the test item is classified as UN GHS category 2.

 

Eye irritation

There is one in vitro eye irritation study available.

 

In the in vitro eye irritation Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), 3 isolated bovine corneas were exposed to Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) in liquid form for 10 minutes using the closed-chamber method. 0.9% sodium chloride was used for the negative control and 1% NaOH solution in highly purified water was used for the positive control. The corneas were rinsed and following a 2-hour post-exposure incubation at 32°C ± 1°C, the opacity and permeability of each cornea was recorded.

 

The positive and negative controls gave the appropriate response (IVIS = 76.999 ± 10.459 and 0.613 ± 1.731, respectively). The mean opacity value for the test substance was 1.992 ± 0.222. The mean permeability OD490 for the test substance was 0.396 ± 0.134. The IVIS for the test substance was 7.927 ± 2.006. The IVIS for Vinyltoluene is > 3 and simultaneously ≤ 55, therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.

Justification for classification or non-classification

Based on the available information in the dossier, the substance Vinyltoluene (CAS No. 25013-15-4) is classified as skin irritation category 2 and no prediction for serious eye damage/eye irritation can be made, when considering the criteria outlined in Annex I of 1272/2008/EC.