Registration Dossier

Administrative data

Description of key information

An in vitro skin corrosion test with MTBAC is available conducted according to OECD 431 guideline and GLP principles, which shows that MTBAC is not corrosive.

An in vivo skin irritation test is available with MTBAC (purity 75%) conducted according to OPPTS guideline and GLP principles, which shows the test item was not irritating. For substance analogue TMAC reliable studies (Klimisch 1 studies) are available that show that TMAC is irritant to the skin. This result is read across to MTBAC.

MTBAC was tested in a BCOP assay, however the outcome precluded conclusions on eye irritancy (result = inconclusive). TBAB and Tributylmethylammoniummethylsulfate are classified for eye irritant properties (cat. 2), based on results from in vivo studies (Klimisch 1 study). This value is read-across to MTBAC.

The rationale to read across these data to MTBAC is attached in IUCLID section 13.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28APR2015 - 01MAY2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted 26 September 2014
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot No.of test material: ZMG-187261
- Expiration date of the lot: 16 April 2016
- Purity test date: 31-07-2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: negative
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Normal, human-derived epidermal keratinocytes cultured to form a multilayered, highly differentiated model of the human epidermis
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: 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 medium.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure (3 minutes): room temperature
- Temperature used during exposure (1 hour) and during post-treatment incubation: 36.4 - 36.7°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: not described
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test substance is decreased below 15%.
- The test substance is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
- 26.0 to 26.7 mg test substance (the skin was moistened with 25 μl Milli-Q water to ensure close contact)
- 50 μl Milli-Q water (negative control)
- 50 μl 8N KOH (positive control)
Duration of treatment / exposure:
3 minutes or 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2/ incubation time
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.4 - 36.7
- Humidity (%): 75 - 87
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 26.0 to 26.7 mg (skin tissue was moistened with 25 μl Milli-Q water to ensure close contact)

VEHICLE
The test substance was \moistened with \ mL of the vehicle.
Duration of treatment / exposure:
3-minute and 1-hour exposure
Number of animals:
2 tissues/ exposure period
Details on study design:
TEST SITE
- EpiDerm Skin Model (EPI-200, Lot no.: 22226 kit N; obtained from MatTek Corporation, Ashland MA, U.S.A). 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.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 3 minutes or one hour

POST INCUBATION PERIOD
- 3 hours

SCORING SYSTEM:
- Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

CONTROLS:
Negative control: 50μL Milli-Q water (Millipore Corp., Bedford, Mass., USA).
Positive control: 50μL 8N KOH.
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minutes incubation
Run / experiment:
1
Value:
86
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
14%
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour incubation
Run / experiment:
1
Value:
83
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
7%
Other effects / acceptance of results:
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with MTBAC compared to the negative control tissues was 86% and 83% respectively. Because the mean relative tissue viability for MTBAC was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment MTBAC is considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 14% and 7% after 1 hour exposure. The maximum inter-tissue variability in viability between two tissues treated identically was less than 22% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%. It was therefore concluded that the test system functioned properly.

MTBAC was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that MTBAC did not interfere with the MTT endpoint.

Interpretation of results:
GHS criteria not met
Conclusions:
An in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that MTBAC is not corrosive in the in vitro skin corrosion test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EpiDerm Skin Model), the influence of MTBAC on the viability of human skin was tested. The test substance was applied directly to 0.6 cm2 cultured skin (26.0 to 26.7 mg, in presence of 25 μl Milli-Q water). After 3 minutes or one hour, the substance was removed and cells were cultured for 3 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 14/% following 3 minute exposure whereas the test substance showed cell viability of 86%. Exposure for 60 minutes resulted in a mean cell viability of 83% and 7% for resp. MTBAC and the positive control. Since the mean relative tissue viability after exposure to MTBAC was above 50% after 3 minute exposure and above 15% after 1 hour exposure, it can be concluded that MTBAC is not corrosive in the in vitro skin corrosion test.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MJV59-115-1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kuiper Rabbitry, Gary, Indiana
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 2.15 to 2.69 kg
- Housing: individually in stainless steel cages
- Diet: Purina Rabbit Chow, ad libitum.
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
7 days
Number of animals:
6
Details on study design:
TEST SITE
- Area of exposure: 6 cm2
- Type of wrap if used: 2.5 cm2 of a two-layer gauze patch held in place with non-irritating Kendall Curity Standard Porous Tape covered with a semi-occlusive plastic overwrap secured in place with Kendall Curity Standard Porous Tape.

REMOVAL OF TEST SUBSTANCE
- The excess test article was removed after exposure
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize scoring system
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animals #1, #3, #4, #5
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 168 hours
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animals #2, # 6
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 168 hours
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Interpretation of results:
GHS criteria not met
Conclusions:
A skin irritation study was conducted in rabbits acording to the OPPTS 870.2500 guideline and in accordance with GLP principles. Effects observed included erythema (max average score 1.33) and edema (max average score 0.33) which were fully reversible within 7 and 2 days, respectively. The test item was concluded to be non-irritant.
Executive summary:

In an OPPTS 870.2500 guideline study (performed in compliance with GLP), the skin irritating potential of the test item was tested in 6 white New Zealand rabbits. 0.5 mL test substance was dermally applied under semi-occlusive conditions. After 4 hours exposure excess test substance was removed. Observations were made for an additional 168 hours after administration. The erythema score (average of 24, 48, and 72 hours) was 1.33 in 2 animals and 1.00 in 4 animals, respectively. Erythema was absent within 7 days after exposure. The edema score (average of 24, 48, and 72 hours) was 0.33 for all six animals. Edema was absent within 48 hours after exposure. The test substance consisted of ca. 75 % of tributylmethylammonium chloride and ca. 25 % water. Effects with the pure substance would likely be more severe.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in section 13.
Reason / purpose:
read-across source
Irritation / corrosion parameter:
% tissue viability
Value:
28
Negative controls validity:
valid
Remarks:
100% viability
Positive controls validity:
valid
Remarks:
5% viability
Other effects / acceptance of results:
The positive control had a mean cell viability after 15 minutes exposure of 46% and did not meet the acceptability criteria. Approximately 1 hour after the performance of this test another in vitro skin irritation test was performed using the same batch of skin and the same batch of 5% (aq) SDS. The positive control had a mean cell viability of 5% and met the acceptability criteria. The absolute mean OD570 of the negative control tissues (in both in vitro skin irritation tests) were within the laboratory historical control data range (See APPENDIX 3). The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. It can be concluded that the deviation in the mean cell viability of the positive control in the first in vitro skin irritation test was caused by a technical error.
The relative mean tissue viability obtained after 15 minutes treatment with TMAC compared to the negative control tissues was 28%. Since the mean relative tissue viability for TMAC was below 50% TMAC is considered to be irritant.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
An in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles with substance analogiue TMAC. It is concluded that this test is valid and that TMAC is irritating in the in vitro skin irritation test. This result is read across to MTBAC.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) according to OECD 439 guideline and GLP principles, the influence of TMAC on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2cultured skin (10.5 to 11.8 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed 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 5% whereas the test substance showed cell viability of 28%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that TMAC is irritating in the in vitro skin irritation test. This result is read across to MTBAC.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For Read Across Justification please refer to Section 13.
Reason / purpose:
read-across source
Irritation parameter:
cornea opacity score
Basis:
animal: #1,#2,#3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal: #1,#2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.33
Max. score:
3
Reversibility:
fully reversible within: 15 days
Irritation parameter:
conjunctivae score
Basis:
animal: #2,#3
Time point:
24/48/72 h
Score:
2.67
Max. score:
3
Reversibility:
fully reversible within: 15 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 8 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 15 days
Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in secton 13.
Reason / purpose:
read-across source
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
24 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
48 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
other: 24, 48, 72 hours and day 7
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
24 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
48 h
Score:
1.67
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
24 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
48 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of 3 rabbits
Time point:
72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
The following were observed in treated rabbits.
Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity was seen in animal no. 1 and 3 whereas scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible was seen in animal no. 2; Area of Opacity- Zero in animal no. 1 and 3 whereas one quarter (or less) but not zero was seen in animal no.2; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in animal no. 2 and some blood vessels definitely hyperaemic (injected) animal no. 1 and 3; Chemosis: Some swelling above normal (includes nictitating membranes) in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3.

Observation at 24 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- Greater than one quarter, but less than half was seen in animal no. 1 and 2 whereas one quarter (or less) but not zero was seen in animal no.3; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no.1 whereas obvious swelling with partial eversion of lids was seen in animal no.2 and 3.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 50%, 60% and 45% damage in animal no. 1, 2 and 3 respectively.

Observation at 48 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in animal no.1 and 3 whereas greater than one quarter, but less than half was seen in animal no. 2; Iris: Normal in all the animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 2 and 3; Chemosis: Obvious swelling with partial eversion of lids was seen in all 3 animals.

Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no. 2.

Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Blood vessels normal was seen in all 3 animals; Chemosis: No swelling (Normal) was seen in all the 3 animals.

The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.33, 0.00, 1.67, 1.33; 1.67, 0.00, 2.00, 2.00; 1.00, and 1.00 0.00, 2.00, 1.67, respectively.

Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity was seen in animal no. 1 and 3 whereas scattered or diffuse areas of opacity (other than slight dulling of normal lustre) details of iris clearly visible was seen in animal no. 2; Area of Opacity- Zero in animal no. 1 and 3 whereas one quarter (or less) but not zero was seen in animal no.2; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in animal no. 2 and some blood vessels definitely hyperaemic (injected) animal no. 1 and 3; Chemosis: Some swelling above normal (includes nictitating membranes) in animal no. 1 and obvious swelling with partial eversion of lids was observed in animal no. 2 and 3.

Observation at 24 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- Greater than one quarter, but less than half was seen in animal no. 1 and 2 whereas one quarter (or less) but not zero was seen in animal no.3; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no.1 whereas obvious swelling with partial eversion of lids was seen in animal no.2 and 3.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 50%, 60% and 45% damage in animal no. 1, 2 and 3 respectively.

Observation at 48 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in animal no.1 and 3 whereas greater than one quarter, but less than half was seen in animal no. 2; Iris: Normal in all the animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal no. 1 whereas diffuse, crimson color; individual vessels not easily discernible was seen in animal no. 2 and 3; Chemosis: Obvious swelling with partial eversion of lids was seen in all 3 animals.

Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was seen in all 3 animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no. 2.
Observation on day 7 after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero percent was seen in all 3 animals; Iris: Normal in all the animals. Conjunctivae - Blood vessels normal was seen in all 3 animals; Chemosis: No swelling (Normal) was seen in all the 3 animals.
No effects were seen in the untreated eyes.
Other effects:
Clinical Observation
No systemic toxicity was observed in treated rabbits during the experimental period.

Mortality
No mortality was observed during the observation period.

Body weight
All rabbits gained weight during experimental period.

Table 1 : Individual Animal Eye Irritation Scores

 

In Treated area Dose:100 mg of test item                                                            Sex:Female

Animal Numbers

1

2

3

Application Side

Left

Right

Right

Eye Reactions

At hour

Day

At hour

Day

At hour

Day

*

1

24

48

72

7

*

1

 24

48

72

7

*

1

24

48

72

7

Corneal Opacity

0

0

1

1

1

0

0

1

1

1

1

0

0

0

1

1

1

0

Area of Opacity

0

0

2

1

1

0

0

1

2

2

1

0

0

0

1

1

1

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctiva

0

2

2

1

2

0

0

1

2

2

2

0

0

1

2

2

2

0

Chemosis

0

1

1

2

1

0

0

2

2

2

2

0

0

2

2

2

1

0

Corneal Damage%

50%

60%

45%

 

Dose:Untreated (Control Eye)                                                                       Sex:Female

Animal Numbers

1

2

3

Application Side

Right

Left

Left

Eye Reactions

At hour

Day

At hour

Day

At hour

Day

*

1

24

48

72

 7

*

1

 24

48

72

7

*

1

24

48

72

7

Corneal Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Area of Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctiva

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Chemosis

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Corneal Damage%

0

0

0

Key:*= Pre-exposure eye examination.

 

Table 1 Continued…

 

Eye Irritation Scores - Mean Values after 24, 48, 72 Hours (Treated eye)

            Animal No.

 Eye Reaction

1

2

3

Corneal Opacity

1.33

1.67

1.00

Iris

0.00

0.00

0.00

Conjunctiva

1.67

2.00

2.00

Chemosis

1.33

2.00

1.67

 

 

Formula :

 

Mean Eye Irritation Score =

                          

Sum of the Individual Animal Score for eye reactionat24, 48 and 72 hours

Number of the Observations (3)

 

  

Table 2 : Individual AnimalClinicalSigns

 

Sex:Female

Animal No.

Days (Post application observation)

0

1

2

3

4

5

6

7

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

3

1

1

1

1

1

1

1

1

Key:1 = Normal

 

Table 3: Individual Animal Body Weight

Sex :Female

Animal No.

Animal Body Weight (kg)

Prior to application

At termination

1

2.208

2.387

2

1.932

2.214

3

2.438

2.646

Key:kg = Kilogram

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the results of an acute in vivo eye irritation study was performed according to OECD guideline 405, tetrabutylammonium bromide is regarded as irritating to eyes (Category 2 classification). This result is read across to MTBAC.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model), the influence of MTBAC on the viability of human skin was tested. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14% following 3 minute exposure whereas the test substance showed cell viability of 86%. Exposure for 60 minutes resulted in a mean cell viability of 83% and 7% for resp. MTBAC and the positive control. Since the mean relative tissue viability after exposure to MTBAC was above 50% after 3 minute exposure and above 15% after 1 hour exposure, it can be concluded that MTBAC is not corrosive in the in vitro skin corrosion test.

In an OPPTS 870.2500 guideline study (performed in compliance with GLP), the skin irritating potential of the test item was tested in 6 white New Zealand rabbits. 0.5 mL test substance was dermally applied under semi-occlusive conditions. After 4 hours exposure excess test substance was removed. Observations were made for an additional 168 hours after administration. The erythema score (average of 24, 48, and 72 hours) was 1.33 in 2 animals and 1.00 in 4 animals, respectively. Erythema was absent within 7 days after exposure. The edema score (average of 24, 48, and 72 hours) was 0.33 for all six animals. Edema was absent within 48 hours after exposure. The test substance consisted of ca. 75 % of tributylmethylammonium chloride and ca. 25 % water. Effects with the pure substance would likely be more severe.

Furthermore, in an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) according to OECD 439 guideline and GLP principles, the influence of TMAC on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2cultured skin (10.5 to 11.8 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed 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 5% whereas the test substance showed cell viability of 28%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that TMAC is irritating in the in vitro skin irritation test. This result is read across to MTBAC.

Eye:

A Bovine Corneal Opacity and Permeability test was conducted according to OECD/EC guidelines and GLP principles. MTBAC induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 8.5 after 240 minutes of treatment. Since MTBAC induced an IVIS > 3 ≤ 55, a prediction on the classification of the substance based on this study only cannot be made.

Based on the results of acute in vivo eye irritation studies according to OECD guideline 405, tetrabutylammonium bromide (TBAB) and Tributylmethylammoniummethylsulfate are regarded as irritating to eyes (Category 2). These results are read across to MTBAC. The rationale to read across these data to MTBAC is attached in IUCLID section 13.

Justification for classification or non-classification

According to the available data, MTBAC is irritating to the skin, but not corrosive and is classified cat.2 according to CLP Regulation (EC) No. 1272/2008. Based on available data, aqueous solutions containing up to and including 75% MTBAC are not classified for skin irritation according to CLP Regulation (EC) No. 1272/2008.

Based on data of a substance analogues, MTBAC was found to be irritating to the eyes and is classified cat. 2 for eye irritation according to CLP Regulation (EC) No. 1272/2008.