Sodium D-glycero-D-gulo-heptonate

Endpoint summary

Description of key information

Skin irritation/corrosion: Two in-vitro studies were conducted to assess the potential of the substance to cause corrosion and irritation. Based on the results of the studies the substance is considered to be non-corrosive to the skin and to be a non-irritant.
Eye irritation: An in-vitro study considered the substance to be a Non-Irritant (NI). In a subsequent in-vivo study iridial inflammation and moderate conjunctival irritation was observed, all of which were fully reversible

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 19 December 2012 and 24 December 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: not applicable in vitro study

Strain:

other: not applicable in vitro study

Details on test animals or test system and environmental conditions:

Not applicable as an in vitro study was conducted.

Type of coverage:

not specified

Preparation of test site:

other: not applicable as in vitro study

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable as in vitro study

Amount / concentration applied:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis. Triplicate tissues treated with 10 μl of DPBS served as the negative controls and triplicate tissues treated with 10 μl of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours

Number of animals:

0 - in vitro study

Details on study design:

PROCEDURE
Pre-Test - Assessment of Direct Test Item Reduction of MTT:
MTT dye metabolism, cell viability assay: The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:

10 mg of the test item was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37ºC, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT.

Pre-Incubation (Day 0: tissue arrival):
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% CO2 in air overnight.

Main Test:
Application of Test Item and Rinsing (Day 1) 2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis. Triplicate tissues treated with 10 μl of DPBS served as the negative controls and triplicate tissues treated with 10 μl of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3):
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30ºC for possible inflammatory mediator determination.

2ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO 2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 μl of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.

For each tissue, duplicate 200 μl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

NEGATIVE AND POSITIVE CONTROL ITEMS
Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control.
Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control

Preparation of Negative and Positive Control Items, MTT and Acidified Isopropanol:
The negative control item was used as supplied.

The positive control item was prepared as a 5% w/v aqueous dilution. A 3 mg/ml MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/ml with assay medium when required. A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.

Irritation / corrosion parameter:

other: other: relative mean viability

Value:

94.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute exposure period followed by a 42 hour post exposure incubation period. Reversibility: other: not classified. Remarks: The relative mean viability of the test item was 94.6% after a 15 minute interval. (migrated information)

Irritant / corrosive response data:

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1. Table 1 can be found below in the attached background information section.

The relative mean viability of the test item treated tissues was 94.6% after a 15-Minute exposure period. This means that the R38 classification is not required for our test material.

Other effects:

Direct MTT Reduction:
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was 7.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.0%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.901 and the standard deviation value of the percentage viability was 6.7%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 7.3%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be Non-Irritant (NI).

Executive summary:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline nonirritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

- OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

- Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results: The relative mean viability of the test item treated tissues was 94.6% after the 15-Minute exposure period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: The test item was considered to be Non-Irritant (NI).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 18 February 2013 and 28 February 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

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

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.36 to 2.57 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml of the test item, which was found to weigh approximately 70 mg (as measured by gently compacting the required volume into an adapted syringe).
- Concentration (if solution): 10% w/w aqueous preparation

Duration of treatment / exposure:

A volume of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released.

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.

Number of animals or in vitro replicates:

Three

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

SCORING SYSTEM: Assessment of ocular damage/irritation was made according to the numerical evaluation, (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).
Any other ocular effects were also noted.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

72974 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects noted.

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

73005 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects noted.

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

73004 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects noted.

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Remarks:

72974 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Remarks:

73005 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Remarks:

73004 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

72974 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

73005 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Effects noted at 1 hour.

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

73004 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Effects noted at 1 hour.

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

72974 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Effects noted at 1 hour.

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

73005 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Effects noted a 1 hour

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

73004 (Male)

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Effects noted at 1 hour

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2 (please see "Any other information on results" below).
No corneal effects were noted during the study.
Iridial inflammation was noted in all treated eyes one hour after treatment.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Minimal conjunctival irritation was noted in all treated eyes at the 24 Hour observation and in two treated eyes at the 48 Hour observation.
One treated eye appeared normal at the 48 Hour observation and two treated eyes appeared normal at the 72 Hour observation.

Other effects:

Bodyweight
One animal showed bodyweight loss and two animals showed expected gain in bodyweight during the study.

Table1              Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	72974Male				73005Male				73004Male
	IPR= 2				IPR = 2				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
IRIS
D	1	0	0	0	1	0	0	0	1	0	0	0
Score (D x 5)	5	0	0	0	5	0	0	0	5	0	0	0
CONJUNCTIVAE
A = Redness	2	1	1	0	2	1	0	0	2	1	1	0
B = Chemosis	1	1	0	0	1	0	0	0	2	0	0	0
C = Discharge	1	0	0	0	2	0	0	0	1	0	0	0
Score (A + B + C) x 2	8	4	2	0	10	2	0	0	10	2	2	0
Total Score	13	4	2	0	15	2	0	0	15	2	2	0

IPR=  Initial pain reaction

Table 2              Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
72974Male	13	4	2	0
73005Male	15	2	0	0
73004Male	15	2	2	0
Group Total	43	8	4	0
Group Mean Score	14.3	2.7	1.3	0.0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced a maximum group mean score of 14.3 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

Introduction. The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method was designed tobe compatible with thefollowing:

- OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted)

- Method B5 Acute Toxicity (Eye Irritation) of CommissionRegulation (EC) No. 440/2008

Result. A single application of the test item to the non-irrigated eye of three rabbits produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 48‑Hour observation and two treated eyes appeared normal at the 72‑Hour observation.

Conclusion. The test item produced a maximum group mean score of 14.3 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

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 validated in-vitro studies were conducted to evaluate the corrosivity potential and skin irritation potential.

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

Introduction:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

The EPISKIN™ model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II &III) test items.

Methods:

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-Ioading. After MTT loading a total biopsy of each epidermis was made a,nd placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 IJI samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (00) was measured at 540 nm (OD540).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results:

The relative mean viability of the test item treated tissues was:

240 minutes exposure: 97.8%

60 minutes exposure: 110.9%

3 minutes exposure: 103.2%

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:

The test item was considered to be Non-Corrosive to the skin.

OECD Guideline 439 (In Vitro Skin Irritation):

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Method:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results:

The relative mean viability of the test item treated tissues was 94.6% after the 15-Minute exposure period.

Quality criteria:

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:

The test item was considered to be Non-Irritant (NI).

Eye Irritation:

In-vitro study:

Introduction.

The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods:

The experimental design of the study consists of a test for direct reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test item for 10 minutes. Triplicate tissues treated with 30 IJI of Solution A served as the negative control and triplicate tissues treated with 30 IJI of 2% w/v Sodium Dodecyl Sulphate (SDS) served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

i) If the percentage relative mean tissue viability was equal or >60% the test item was considered to be non-irritant (NI).

ii) If the percentage relative mean tissue viability was < 60% the test item was considerd to be an irritant (I)

Results.

The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 98.6%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criterion.

The quality criterion required for acceptance of results in the test was satisfied.

Conclusion:

According to the study plan followed the test item was considered to be a Non-Irritant (NI).

In-vivo study:

Introduction. 

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method was designed tobe compatible with thefollowing:

- OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted)

- Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008

Result. 

A single application of the test item to the non-irrigated eye of three rabbits produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 48‑Hour observation and two treated eyes appeared normal at the 72‑Hour observation.

Conclusion. 

The test item produced a maximum group mean score of 14.3 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for selection of skin irritation / corrosion endpoint:
This study has been conducted according to GLP and according to OECD guidelines. Due to these reasons the study has been assigned a klimisch reliability rating of 1 according to the Klimisch scale.

Justification for selection of eye irritation endpoint:
This study has been conducted according to GLP and according to OECD guidelines. Due to these reasons the study has been assigned a klimisch reliability rating of 1 according to the Klimisch scale.

Justification for classification or non-classification

Skin irritation/corrosion:

The substance is not classified as an irritant or corrosive, based on the results of two in-vitro studies conducted, which gave the following results:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test): The test item was considered to be Non-Corrosive to the skin.

OECD Guideline 439 (In Vitro Skin Irritation): The test item was considered to be Non-Irritant (NI).

Both these in-vitro methods have been validated and accepted and are considered appropriate for classification purposes.

Eye irritation:

The results from the eye irritation study were evaluated according to the Classification, Labelling and Packaging Regulation (CLP) criteria and Directive 67/548/EEC (DSD) criteria.

The mean scores of individual animals for corneal opacity, iritis, conjunctival redness and chemosis (based on gradings at 24, 48 and 72 hours) were calculated for each of the three animals tested. There was a maximum mean score of 0.66 for conjunctival redness (in two animals) and a maximum mean score of 0.33 for chemosis (in one animal). The scores for iritis and corneal opacity were 0 for all animals.

The mean scores for redness of 0.66 were below the threshold values for a positive response (≥2 for redness under CLP and≥2.5 for redness under DSD). The mean score for chemosis of 0.33 was below the threshold value for a positive response (≥2 under CLP and DSD).

All effects were fully reversible.

The substance therefore does not meet the criteria for classification as an eye irritant according to either the CLP or DSD criteria.