Magnesium octadecylbenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-May-2014 to 26-May-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10.2 to 10.9 mg, moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

Exposure:15 minutes
Post incubation period: 42 hours

Details on study design:

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Irritation / corrosion parameter:

other: other: percentage viability

Value:

120

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. (migrated information)

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.

It is concluded that this test is valid and that EXP1313100 is non-irritant in the in vitro skin irritation test.

Executive summary:

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 EXP1313100 compared to the negative control tissues was 120%. Since the mean relative tissue viability for EXP1313100 was above 50% after 15 minutes treatment EXP1313100 is considered to be non-irritant.

 

The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 June 2016 to 06 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., St. Constant, Quebec, Canada.
- Age at study initiation: 16 weeks
- Weight at study initiation: 2.4 to 2.6 kg
- Housing: The animals were single housed in stainless steel cages equipped with an automatic watering valve as specified in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals from the National Research Council.1 Each cage was cl arly labeled with a color-coded cage card indicating study, group, animal number, and sex.
- Diet (e.g. ad libitum): PMI Nutrition International Certified Rabbit Chow No. 5322 was provided ad libitum throughout the study except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely
available to each animal via an automatic watering system, except during designated procedures.
- Acclimation period: The animals were acclimated to their designated housing for 7 days before the first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 23°C)
- Humidity (%): 48% to 60%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle was maintained, except when interrupted for
designated procedures.

IN-LIFE DATES: From: To: 21 June 2016 to 06 July 2016

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 g moistened with 0.5 mL of water.

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: inch x 1 inch square
- Type of wrap if used: The gauze patch was held in contact with the skin at the cut edges with a nonirritating tape. Removal and ingestion of the test substance were prevented by placing an elastic wrap over the trunk and test area (semi-occlusive binding). The elastic wrap was then further secured with adhesive tape around the trunk at the cranial and caudal ends. After dosing, collars were placed on each animal and remained in place until removal on Day 3.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The residual test substance was removed using gauze moistened with RODI water followed by dry gauze.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1, 24, 48, and 72 hours and up to 14 days after patch removal

SCORING SYSTEM:
- Method of calculation: Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks:

Grade 1 edema recorded on days 4, 5 and 6

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Exposure to the test substance produced well-defined erythema at 3/3 test sites by the 1-hour scoring interval. The well-defined erythema persisted through study completion on Day 14 for Animal No. 5553 and Animal No. 5554, with the exception of very slight erythema that was noted on Day 7 and Days 6, 9, and 10, respectively. Animal No. 5555 had well-defined erythema from the 1-hour scoring interval through Day 6, very slight erythema from Day 6 to Day 8, well-defined erythema on Day 9, and then very slight erythema from Day 10 through study completion on Day 14. Erythema was present for all 3 animals at the time of study completion.

Very slight edema occurred at 2/3 test sites by the 24-hour interval, increased to slight edema by the 48-hour interval, and returned to very slight edema at the 72-hour scoring interval. The edema continued through Day 6 for 2/3 test sites, with the other 1/3 test sites showing very slight edema from Day 4 to Day 6. All edema resolved by Day 7.

Focal and/or pinpoint (up to 10% of the test site) blanching was observed for Animal No. 5555 from the 72-hour scoring interval through Day 9 and for Animal No. 5554 on Days 5 to 7 and 9 to 10. Desquamation of the test site was noted for Animal No. 5555 from Day 10 through study completion on Day 14.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of the test (4-hour exposure), the registration substance is considered to be an irritant (Category 2) according to the GHS Classification and Labelling System, due to the persistence of inflammation throughout the 14 day observation period.

Executive summary:

The objective of this study was to assess the irritant and/or corrosive effects of the registration substance when given as a single dermal administration to rabbits. Each of 3 rabbits received a 0.5 g dose of the test substance as a single dermal application. The dose was held in contact with the skin under a semi-occlusive binder for an exposure period of 4 hours. Following completion of the exposure period, the binder was removed and the remaining test substance was wiped from the skin using gauze moistened with reverse osmosis deionized (RODI) water followed by dry gauze. Test sites were subsequently examined and scored for dermal irritation up to Day 14.

Exposure to the test substance produced well-defined erythema at 3/3 test sites by the 1-hour scoring interval. The well-defined erythema persisted through study completion on Day 14 for Animal No. 5553 and Animal No. 5554, with the exception of very slight erythema that was noted on Day 7 and Days 6, 9, and 10, respectively. Animal No. 5555 had well-defined erythema from the 1-hour scoring interval through Day 6, very slight erythema from Day 6 to Day 8, well-defined erythema on Day 9, and then very slight erythema from Day 10 through study completion on Day 14. Erythema was present for all 3 animals at the time of study completion.

Very slight edema occurred at 2/3 test sites by the 24-hour interval, increased to slight edema by the 48-hour interval, and returned to very slight edema at the 72-hour scoring interval. The edema continued through Day 6 for 2/3 test sites, with the other 1/3 test sites showing very slight edema from Day 4 to Day 6. All edema resolved by Day 7.

Under the conditions of the test (4-hour exposure),EXP1313100 is considered to be an irritant (Category 2) according to the GHS Classification and Labelling System.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

key study

Study period:

19-May-2014 to 26-May-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10.2 to 10.9 mg, moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

Exposure:15 minutes
Post incubation period: 42 hours

Details on study design:

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Irritation / corrosion parameter:

other: other: percentage viability

Value:

120

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. (migrated information)

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.

It is concluded that this test is valid and that EXP1313100 is non-irritant in the in vitro skin irritation test.

Executive summary:

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 EXP1313100 compared to the negative control tissues was 120%. Since the mean relative tissue viability for EXP1313100 was above 50% after 15 minutes treatment EXP1313100 is considered to be non-irritant.

 

The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

09 June 2016 to 06 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., St. Constant, Quebec, Canada.
- Age at study initiation: 16 weeks
- Weight at study initiation: 2.4 to 2.6 kg
- Housing: The animals were single housed in stainless steel cages equipped with an automatic watering valve as specified in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals from the National Research Council.1 Each cage was cl arly labeled with a color-coded cage card indicating study, group, animal number, and sex.
- Diet (e.g. ad libitum): PMI Nutrition International Certified Rabbit Chow No. 5322 was provided ad libitum throughout the study except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely
available to each animal via an automatic watering system, except during designated procedures.
- Acclimation period: The animals were acclimated to their designated housing for 7 days before the first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 23°C)
- Humidity (%): 48% to 60%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle was maintained, except when interrupted for
designated procedures.

IN-LIFE DATES: From: To: 21 June 2016 to 06 July 2016

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 g moistened with 0.5 mL of water.

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: inch x 1 inch square
- Type of wrap if used: The gauze patch was held in contact with the skin at the cut edges with a nonirritating tape. Removal and ingestion of the test substance were prevented by placing an elastic wrap over the trunk and test area (semi-occlusive binding). The elastic wrap was then further secured with adhesive tape around the trunk at the cranial and caudal ends. After dosing, collars were placed on each animal and remained in place until removal on Day 3.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The residual test substance was removed using gauze moistened with RODI water followed by dry gauze.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1, 24, 48, and 72 hours and up to 14 days after patch removal

SCORING SYSTEM:
- Method of calculation: Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks:

Grade 1 edema recorded on days 4, 5 and 6

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Exposure to the test substance produced well-defined erythema at 3/3 test sites by the 1-hour scoring interval. The well-defined erythema persisted through study completion on Day 14 for Animal No. 5553 and Animal No. 5554, with the exception of very slight erythema that was noted on Day 7 and Days 6, 9, and 10, respectively. Animal No. 5555 had well-defined erythema from the 1-hour scoring interval through Day 6, very slight erythema from Day 6 to Day 8, well-defined erythema on Day 9, and then very slight erythema from Day 10 through study completion on Day 14. Erythema was present for all 3 animals at the time of study completion.

Very slight edema occurred at 2/3 test sites by the 24-hour interval, increased to slight edema by the 48-hour interval, and returned to very slight edema at the 72-hour scoring interval. The edema continued through Day 6 for 2/3 test sites, with the other 1/3 test sites showing very slight edema from Day 4 to Day 6. All edema resolved by Day 7.

Focal and/or pinpoint (up to 10% of the test site) blanching was observed for Animal No. 5555 from the 72-hour scoring interval through Day 9 and for Animal No. 5554 on Days 5 to 7 and 9 to 10. Desquamation of the test site was noted for Animal No. 5555 from Day 10 through study completion on Day 14.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of the test (4-hour exposure), the registration substance is considered to be an irritant (Category 2) according to the GHS Classification and Labelling System, due to the persistence of inflammation throughout the 14 day observation period.

Executive summary:

The objective of this study was to assess the irritant and/or corrosive effects of the registration substance when given as a single dermal administration to rabbits. Each of 3 rabbits received a 0.5 g dose of the test substance as a single dermal application. The dose was held in contact with the skin under a semi-occlusive binder for an exposure period of 4 hours. Following completion of the exposure period, the binder was removed and the remaining test substance was wiped from the skin using gauze moistened with reverse osmosis deionized (RODI) water followed by dry gauze. Test sites were subsequently examined and scored for dermal irritation up to Day 14.

Exposure to the test substance produced well-defined erythema at 3/3 test sites by the 1-hour scoring interval. The well-defined erythema persisted through study completion on Day 14 for Animal No. 5553 and Animal No. 5554, with the exception of very slight erythema that was noted on Day 7 and Days 6, 9, and 10, respectively. Animal No. 5555 had well-defined erythema from the 1-hour scoring interval through Day 6, very slight erythema from Day 6 to Day 8, well-defined erythema on Day 9, and then very slight erythema from Day 10 through study completion on Day 14. Erythema was present for all 3 animals at the time of study completion.

Very slight edema occurred at 2/3 test sites by the 24-hour interval, increased to slight edema by the 48-hour interval, and returned to very slight edema at the 72-hour scoring interval. The edema continued through Day 6 for 2/3 test sites, with the other 1/3 test sites showing very slight edema from Day 4 to Day 6. All edema resolved by Day 7.

Under the conditions of the test (4-hour exposure),EXP1313100 is considered to be an irritant (Category 2) according to the GHS Classification and Labelling System.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-May-2014 to 28-May-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 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”

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the test substance was tested neat via a gauze patch approximately 2cm2.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 10% (w/v) Benzalkonium Chloride

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours

Details on study design:

TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
IVIS score:
= 3 No Category
> 3; = 55 No prediction can be made
>55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

3.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

5.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Since EXP1313100 induced an IVIS > 3 = 55, no prediction on the classification can be made.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Since EXP1313100 induced an IVIS > 3 = 55, no prediction on the classification can be made.

Executive summary:

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 126 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

EXP1313100 induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 4.3 after 10 minutes of treatment.

.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 25 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Animals used within the study were 12 - 24 weeks old.
- Weight at study initiation: Body weights were between 2632 - 2860 kg.
- Housing: Individually housed in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 15 - 25 December 2014

Vehicle:

other:

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit):
0.1 mL of the test substance formulation

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 50% (w/w) dilution in propylene glycol. The formulations were kept at room temperature and dosed within 4.5 hours after adding the vehicle to the test substance. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance. Homogeneity was obtained to visually acceptable levels and the formulations were stirred during dosing, which ensured homogeneity sufficient for these kinds of studies. In order to obtain homogeneity, the test substance formulations were heated in a water bath with a maximum temperature of 96.4 ºC for a maximum of 45 minutes. The test substance formulations were allowed to cool down to a temperature of maximally 26.4 ºC prior to dosing.

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance

Number of animals or in vitro replicates:

3 males

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner one week later, after considering the degree of eye irritation observed in the first animal.

PREEMPTIVE PAIN MANAGEMENT
One hour prior to instillation of the test substance, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test substance, two drops of the topical anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.

TREATMENT
Each animal was treated by instillation of 0.1 mL of the test substance formulation, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control. Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection. After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system according to OECD 405.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritant / corrosive response data:

Irritation: Instillation of 0.1 mL of the test material into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 24 hours in one animal and within 48 hours in the other two animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Corrosion: There was no evidence of ocular corrosion.

Other effects:

Colouration/Remnants: Remnants of the test substance noted for all animals on the outside of the eyelids on Day 1.

Toxicity/Mortality: No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results, the test substance does not have to be classified and has no obligatory labelling requirement for eye irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments);
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

key study

Study period:

13-May-2014 to 28-May-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: 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”

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the test substance was tested neat via a gauze patch approximately 2cm2.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 10% (w/v) Benzalkonium Chloride

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours

Details on study design:

TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
IVIS score:
= 3 No Category
> 3; = 55 No prediction can be made
>55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

3.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

5.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Since EXP1313100 induced an IVIS > 3 = 55, no prediction on the classification can be made.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Since EXP1313100 induced an IVIS > 3 = 55, no prediction on the classification can be made.

Executive summary:

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 126 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

EXP1313100 induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 4.3 after 10 minutes of treatment.

.

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

15 - 25 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Animals used within the study were 12 - 24 weeks old.
- Weight at study initiation: Body weights were between 2632 - 2860 kg.
- Housing: Individually housed in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 15 - 25 December 2014

Vehicle:

other:

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit):
0.1 mL of the test substance formulation

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 50% (w/w) dilution in propylene glycol. The formulations were kept at room temperature and dosed within 4.5 hours after adding the vehicle to the test substance. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance. Homogeneity was obtained to visually acceptable levels and the formulations were stirred during dosing, which ensured homogeneity sufficient for these kinds of studies. In order to obtain homogeneity, the test substance formulations were heated in a water bath with a maximum temperature of 96.4 ºC for a maximum of 45 minutes. The test substance formulations were allowed to cool down to a temperature of maximally 26.4 ºC prior to dosing.

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance

Number of animals or in vitro replicates:

3 males

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner one week later, after considering the degree of eye irritation observed in the first animal.

PREEMPTIVE PAIN MANAGEMENT
One hour prior to instillation of the test substance, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test substance, two drops of the topical anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.

TREATMENT
Each animal was treated by instillation of 0.1 mL of the test substance formulation, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control. Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection. After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system according to OECD 405.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritant / corrosive response data:

Irritation: Instillation of 0.1 mL of the test material into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 24 hours in one animal and within 48 hours in the other two animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Corrosion: There was no evidence of ocular corrosion.

Other effects:

Colouration/Remnants: Remnants of the test substance noted for all animals on the outside of the eyelids on Day 1.

Toxicity/Mortality: No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results, the test substance does not have to be classified and has no obligatory labelling requirement for eye irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments);
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The overall weight of evidence indicates that alkaryl sulfonates like that of the target substance to not possess significant skin irritant properties when tested in solvent oil, which is most relevant form to test in given the use sector of these substances as a lubricant additive. When tested in the absence of solvent oil the substances cause mild irritancy, which may warrant classification if the irritancy persists for the duration of the study.

The data on the source substance indicates there is no significant eye irritancy potential, therefore it is concluded that the target substance doesn't meet the classification criteria for eye irritancy.