Short Chain Fructo Oligosaccharides

Endpoint summary

Description of key information

Irritation/corrosion studies have been conducted with ScFOS.

Thus:

- For skin irritation, the ScFOS is not irritating on skin (OECD 431 and OECD 439).

- For eye irritation, the ScFOS is not irritating on eyes (OECD 438).

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:

June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU method B.40bis

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

• Sponsor’s identification: ACTILIGHT® 950P
• Form: powder
• Colour: White
• Storage : room temperature

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epidermal Skin Test EST-1000, Cell Systems
- Tissue batch number(s): - Batch No. EST- 110502-001)

The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which has been previously filled with 1 mL of maintenance medium (Cell Systems MM-160511). The culture dishes were incubated at 37°C, 5% CO2 during 20 hours and 25 minutes before treatment.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room température
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
3 minutes and 1 hour after the test item application, the human epidermis was washed 20 times with 1 mL of PBS (PAN BIOTECH GmbH, Batch No. 4721010).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration / Incubation time: The skin sample was placed in MTT solution of 1 mg/mL concentration for 3 hours at 37°C, 5% CO2.
The precipitated blue formazan product is then extracted using isopropanol during 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extraction in isopropanol (1:3).
The absorbance was measured in triplicate of MTT extract.
The measured absorbances are proportional to the number of live cells.

The direct interaction of MTT with the test item was checked by adding 25 mg of ACTILIGHT® 950P to 0.3 mL of the solution of MTT at 1 mg/mL. No blue or purple coloration was noted after 3 hours of incubation at 37°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is striçtly less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if 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:
The test item has been applied, as supplied, at the dose of 25 mg, to the epidermal surface of 2 human skin models, then 25 µL pf PBS was added.


NEGATIVE CONTROL
In the same experimental conditions, a negative control (PBS ) has been carried out.

POSITIVE CONTROL
In the same experimental conditions, a positive control (8N KOH) has been carried out.

Duration of treatment / exposure:

The test item remained on the epidermis during 3 minutes and during 1 hour at room température

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

115.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1hour

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct-MTT reduction:
The direct interaction of MTT with the test item was checked by adding 25 mg of ACTILIGHT® 950P to 0.3 mL of the solution of MTT at 1 mg/mL. No blue or purple coloration was noted after 3 hours of incubation at 37°C, 5% CO2.
Therefore, there is no direct interaction between the test item and MTT

Interpretation of results:

GHS criteria not met

Conclusions:

under the conditions of the test, 3 minutes and 1 hour after the test item application, the mean viability of the epidermis skins treated with the test item was 115.6% (considered as 100%) and 97.3%, versus 2.7% and 1.0%, respectively, in the positive control (potassium hydroxide 8N). The substance is considered as not corrosive to the skin in accordance with the Regulation EC No. 1272/2008, the test item must not be classified in category 1 “Corrosive”.

Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (Epidermal Skin Test EST-1000, Cell Systems). The experimental protocol was established in accordance with the OECDguideline No 431 dated April 13th, 2004 and the method B.40bis of the Council regulation No. 440/2008.

The test item ACTILIGHT® 950P was applied, as supplied, at the dose of 25 mg, to 2 Human skin model surfaces (Epidermal Skin Test EST-1000, Cell Systems), then 25 µL of PBS was added to ensure a good contact with the epidermis during 3 minutes and 1 hour.

 

3 minutes and 1 hour after the test item application, the mean viability of the epidermis skins treated with the test item was 115.6% (considered as 100%) and 97.3%, versus 2.7% and 1.0%, respectively, in the positive control (potassium hydroxide 8N).

 

The results obtained, under these experimental conditions, enable to conclude that the test item must not be classified in category 1 “Corrosive” in accordance with the Regulation EC No. 1272/2008. The substance is considered a not corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2011

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

• Sponsor’s identification: ACTILIGHT® 950P
• Form: powder
• Colour: White

Test system:

human skin model

Remarks:

Episkin Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epsiskin model

Vehicle:

unchanged (no vehicle)

Details on test system:

1) Human skin model:
The 0.38 cm2 reconstituted epidermis (Episkin) were received on 07 June 2011. The insert (filter + epidermis) was gently removed from the agarose while avoiding leasing agarose on the polycarbonate filter. The inserts were placed in a 12 wells culture plated which has been previously filled with 2.2 mL of maintenance medium (Skinethic).

2) Treatment:
After a maintenance period of 4 hours, the inserts were placed in a 12 wells culture plated which has been previously filled with 2.2 mL of assay medium (Skinethic).
The test item was applied, as supplied, at the dose of 10 mg, to the epidermal surface of 3 human skin models, previously moistened with 5 µL of distilled water, during 15 minutes in an incubator at 37°C, 5% CO2.

In the same experimental conditions, a positive control (5% SDS, CAS No. 151-21-3,) and a negative control (distilled water ) were carried out.

3) Grading of reactions
15 minutes after the test item application, the human epidermis was washed with PBS (3 x 0.5 mL) and was incubated for a 42 hours post-treatment incubation period of the rinsed tissues in fresh medium at 37°C, 5% CO2. Then, the epidermis was put in contact with the MTT solution.

The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazo1-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal. The skin sample was placed in MTT solution of 0.5 mg/mL concentration for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product is then extracted using acidic isopropanol during 2 hours and 10 minutes under agitation in the dark, and the concentration of formazan was measured by deteimining the Optical Density (OD) at 570 nm.
The absorbance was measured in triplicate of MTT extract.
The measured absorbances are proportional to the number of live cells.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin
- Tissue batch number(s): batch No. 11-EKIN-023
- Delivery date: 07 June 2011
- Date of initiation of testing: 07 June 2011 and 09 June 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation : 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 x 0.5 ml

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution of 0.5 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Wavelength: 570 nm.

PREDICTION MODEL / DECISION CRITERIA:
The optical denslty (OD) values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%.
- The test substance is considered to be non irritant to skin if : the viability after 15 minutes of exposure and 42 hours of post-treatment incubation is > 50%.
- The test substance is considered to be irritant to skin if: the tissue viability after 15 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.


P

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg of pure test ltem on 0.38 cm2 human skin model

Duration of treatment / exposure:

15 minutes in an incubator at 37°C, 5% CO2

Duration of post-treatment incubation (if applicable):

42 hours post-treatment incubation.

Number of replicates:

3 human skin models per

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of the 3 replicates

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

10.7%

Other effects / acceptance of results:

The direct interaction of MTT with the test item was checked by adding 10 mg of ACTILIGHT‘950P to 2.2 mL of the solution of MTT at 0.5 mg'mL. No blue or purple coloration was noted after 3 hours of incubation at 37°C, 5% CO2,. Therefore, there is no direct interaction between the-test item and MTT.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the mean viability of the epidermis skins was 109.9% (considered as 100%), versus 10.7% in the positive control (5% Sodium Dodecyl Sulfate).
The substance is considered as not irritating to the skin in accordance with the Regulation EC No. 1272/2008.

Executive summary:

The aim of the study was to evaluate the possible irritating effects of ACTILIGHT 950P after topical administration on in vitro human reconstructed epidermis (Episkin model).

The test item ACTILIGHT 950P was applied, as supplied, at the dose of 10 mg, to 3 Human skin model surfaces (Episkin ) moistened with 5 µL of distilled water, during 15 minutes, followed by a 42 hours post-incubation period at 37°C, 5% CO . The experimental protocol was established in accordance with OECD guideline No. 439 adopted 22 July 2010 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

 

The mean viability of the epidermis skins was 109.9% (considered as 100%), versus 10.7% in the positive control (5% Sodium Dodecyl Sulfate).

 

The results obtained, under these experimental conditions, enable to conclude that the test item ACTILIGHT 950P must not be classified in accordance with the Regulation EC No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

• Sponsor’s identification: ACTILIGHT® 950P
• Form: powder
• Colour: White
• Storage : room temperature

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight):
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples:
- Indication of any antibiotics used:

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test item was applied, as supplied, to the cornea, such that the entire surface of the cornea is evenly covered with the test item.

Duration of treatment / exposure:

The test item was applied for 10 seconds

Number of animals or in vitro replicates:

to 3 enucleated chicken eyes

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Source, collection and transport of chicken eyes:
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).

Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 19 May 2011 at 8:30 am.

Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient tetnperature in polystyrene boxes humidified with toz'els moistened with isotonic saline.
The eyes were enucleated at Phycher on 19 May 2011 at 9:40 am


Preparation of eyes:
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the isotonic saline drip. The Ghambers of the superfusion apparatus was temperature controlled at 32°C 1.5°C.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% fi om the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes have been examined and approved, the eyes were incubated between 60 and 76 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time= 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES : to 3 enucleated chicken eyes

APPLICATION DOSE AND EXPOSURE TIME AND REMOVAL OF TEST SUBSTANCE:
Test item:
Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied, to the cornea, such that the entire surface of the cornea is evenly covered with the test item.

The test item was applied for 10 seconds and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

Controls:
Concurrent negative control (physiological saline — Cooper, Batch No. 19OC31GE) and positive control (sodium hydroxide — Sigma, Batch No. MKBF9973V) were included in this experiment.

METHODS FOR MEASURED ENDPOINTS:
Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (d 5 minutes) after the post-treatment rinse.
The endpoints evaluated are corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of ftuorescein retention (which is determined only at pretreatment and 30 minutes after test item exposure) are determined at each of the above time points.
The eyes were not kept in an appropriate fixative at the end of the study for possible histopathological examination.

Irritation parameter:

cornea opacity score

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as corrosive/severe irritant, as expected.

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as non corrosive/severe irritant, as expected.

Results from corneal opacity, swelling, and fluorescein retention are evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.

Once each endpoint has been evaluated, ICE classes can be assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes is done according to the scales of the OECD 438 guideline.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the ocular reactions observed in eyes treated with the test item were slight to moderate:
- maximal mean score of corneal opacity: 1.5, corresponding to the ICE class II;
- mean score of fluorescein retention: 1.2, corresponding to the ICE class II;
-maximal mean corneal swelling: +4%, corresponding to the ICE class I.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item ACTILIGHT 950P was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes 1'ere rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The experimental protocol was established in accordance with the OECD guideline No 438 adopted 07 September 2009 and the test method B.48 — Commission Regulation (EU) No. 1152/2010 dated 08 December 2010.

The ocular reactions observed in eyes treated with the test item were slight to moderate:

- maximal mean score of corneal opacity: 1.5, corresponding to the ICE class II;

- mean score of fluorescein retention: 1.2, corresponding to the ICE class II;

-maximal mean corneal swelling: +4%, corresponding to the ICE class I.

 

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as corrosive/severe irritant, as expected.

 

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as non corrosive/severe irritant, as expected.

 

The results obtained, under these experimental conditions, enable to conclude that the test item ACTILIGHT 950P must not be classified in category 1 “irreversible effects on the eye in accordance with the Regulation (EC) No. 1272/2008.

 

Furthermore, according to the OECD guideline 438 adopted the 25 june 2018 when the combinations of the 3 endpoints is 3 x I, 2 x I, 1 x II or 2 x II, 1 x I, no UN GHS classification is needed. Therefore, test item ACTILIGHT 950P is not requiring classification (UN GHS classification category)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

Two in vitro studies indicate no skin irritant effects for ScFOS

An in vitro study performed according to the OECD 431 was conducted (GLP compliant) (Colas 2011).  In this study, the test item scFOS was applied, as supplied, at the dose of 25 mg, to 2 Human skin model surfaces (Epidermal Skin Test EST-1000, Cell Systems), then 25 µL of PBS was added to ensure a good contact with the epidermis during 3 minutes and 1 hour. 3 minutes and 1 hour after the test item application, the mean viability of the epidermis skins treated with the test item was 115.6% (considered as 100%) and 97.3%, versus 2.7% and 1.0%, respectively, in the positive control (potassium hydroxide 8N). The ScFOS is considered a not corrosive to the skin.

An in vitro study performed according to the OECD 439 was also conducted (GLP compliant) (Colas 2011).  In this study, the test item ScFOS was applied, as supplied, at the dose of 10 mg, to 3 Human skin model surfaces (Episkin ) moistened with 5 µL of distilled water, during 15 minutes, followed by a 42 hours post-incubation period at 37°C, 5% CO. The mean viability of the epidermis skins was 109.9% (considered as 100%), versus 10.7% in the positive control (5% Sodium Dodecyl Sulfate). The scFOS is considered a not irritant to the skin.

Eye irritation:

An in vitro study performed according to the OECD 438 was conducted (GLP compliant) (Colas 2011).  In this study, the test item ScFOS was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. The ocular reactions observed in eyes treated with the test item were slight to moderate:

- maximal mean score of corneal opacity: 1.5, corresponding to the ICE class II;

- mean score of fluorescein retention: 1.2, corresponding to the ICE class II;

- maximal mean corneal swelling: +4%, corresponding to the ICE class I.

The results obtained, under these experimental conditions, enable to conclude that the ScFOS must not be classified in category 1 “irreversible effects on the eye in accordance with the Regulation (EC) No. 1272/2008.

Furthermore, according to the OECD guideline 438 adopted the 25 june 2018 when the combinations of the 3 endpoints is 3 x I, 2 x I, 1 x II or 2 x II, 1 x I, no UN GHS classification is needed. Therefore, ScFOS is not requiring classification (UN GHS classification category).

 

Testing wers conducted according to a scientifically valid and accepted methods. Hence, the obtained results can be considered as reliable.

Justification for classification or non-classification

Skin irritation:

No classification is proposed. There is no evidence from in vitro studies that scFOS is a skin irritant.

Eye irritation:

No classification is proposed. There is no evidence from in vitro studies that scFOS is an eye irritant.