Iron orthophosphate

Endpoint summary

Description of key information

Skin irritation: OECD 439, RL1: not irritating

Skin corrosion: OECD 431, RL1: not corrosive

Eye irritation: OECD 405, RL1: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 15 November 2011 and 18 November 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)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Study designed to be compatible with this guideline, however the guideline is not referred to in the study report.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN(TM) Model Kit 0.38 cm² (SkinEthic Laboratories, Nice, France)
- Tissue batch number(s): not reported
- Production date: not reported
- Shipping date: not reported
- Delivery date: 15 November 2011
- Date of initiation of testing: 09 November 2011

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without reference filter

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive (H314, Category 1A) to skin if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive (H314, Category 1B) to skin if the viability after 3 minutes exposure is greater or equal to 35% or if the viability after 60 minutes exposure is less than 35%.
- The test substance is considered to be corrosive (H314, Category 1C) to skin if the viability after 60 minutes exposure is greater or equal to 35%, or if the viability after 240 minutes exposure is less than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 240 minutes exposure is greater than or equal to 35%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg (100 μL of 0.9% w/v sodium chloride solution was added for wetting of the test item.)

NEGATIVE CONTROL
- Amount(s) applied: 50µL
- Concentration: 0.9% w/v

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3, 60 & 240 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

110.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

96.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minute exposure

Value:

87.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:

RESULTS

Direct MTT Reduction

The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

Test Item, Positive Control Item and Negative Control Item

Mean OD₅₄₀ values and viabilities for the negative control, positive control item and test item are given in Table 1.

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

240 minutes exposure                      :          87.1 %

60 minutes exposure                        :          96.4 %

3 minutes exposure                          :          110.8 %

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 3.1 % relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Period	Mean OD540of duplicate tissues	Relative mean viability (%)
Negative Control Item	240 Minutes	0.194	100*
Positive Control Item	240 Minutes	0.006	3.1
Test Item	240 Minutes	0.169	87.1
	60 Minutes	0.187	96.4
	3 Minutes	0.215	110.8

*=     The mean viability of the negative control tissues is set at 100

Interpretation of results:

other: non-corrosive

Conclusions:

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

Executive summary:

Introduction: The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKIN^TM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with the following:

• OECD Guideline for the Testing of Chemicals No. 431 “In VitroSkin Corrosion: Human Skin Model Test” (adopted 13 April 2004)
• Method B.40 of CommissionRegulation (EC) No. 440/2008

The EPISKIN^TM 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) chemicals.

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-loading. 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 (OD) 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	:	87.1%
60 minutes exposure	:	96.4%
3 minutes exposure	:	110.8%

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 15 November 2011 and 21 November 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)

Version / remarks:

22 July 2010

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)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN (SkinEthic Laboratories, Nice, France)
- Tissue batch number(s): not reported
- Production date: not reported
- Shipping date: not reported
- Delivery date: 15 November 2011 (as stated in the study report)
- Date of initiation of testing: 09 November 2011 (as stated in the study report)

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: 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.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported


NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability after 15 minutes exposure is greater than 50%.
- The test substance is considered to be irritant (H315, Category 2) to skin if the relative mean tissue viability after 15 minutes exposure is less than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg (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.)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL DPBS

POSITIVE CONTROL
- Amount(s) applied: 10 µL SDS
- Concentration: 5%

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean out of 3

Value:

104.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

RESULTS

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.

Test Item, Positive Control Item and Negative Control Item

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.

The relative mean viability of the test item treated tissues was 104.1% after a 15-Minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 6.0% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.8%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was 0.938 and the standard deviation value of the percentage viability was 6.8%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 3.7%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540of tissues	Mean OD540of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.947	0.938	0.064	101.0	100*	6.8
	0.870			92.8
	0.997			106.3
Positive Control Item	0.068	0.057	0.017	7.2	6.0	1.8
	0.037			3.9
	0.065			6.9
Test Item	0.989	0.976	0.035	105.4	104.1	3.7
	1.003			106.9
	0.937			99.9

SD=    Standard deviation

*=     The mean viability of the negative control tissues is set at 100%

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulations (EC) No 1272/2008

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^TM 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. 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 104.1% 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:

Between 20 December 2011 and 07 January 2012

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

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Two 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 2.57 or 2.86 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.
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:

yes, concurrent no treatment

Amount / concentration applied:

A volume of 0.1 mL of the test item, which was found to weigh approximately 94 mg

Duration of treatment / exposure:

Up to 1 hour

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

2 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, an additional animal was treated. )

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.
Initially, a single rabbit was treated. A volume of 0.1 ml of the test item, which was found to weigh approximately 94 mg (as measured by gently compacting the required volume into an adapted syringe) 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. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 1.
After consideration of the ocular responses produced in the first treated animal, a second animal was treated. In order to minimise pain on application of the test item, one drop of local anaesthetic (Tetracaine hydrochloride 0.5%, Bausch & Lomb, Surrey, UK) was instilled into both eyes of the second animal 1 to 2 minutes before treatment.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (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. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

cornea opacity score

Basis:

animal: 71491 Male

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Basis:

animal: 71516 Male

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal: 71491 Male

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effects observed

Irritation parameter:

iris score

Basis:

animal: 71516 Male

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Irritation parameter:

conjunctivae score

Basis:

animal: 71491 Male

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal: 71516 Male

Time point:

24/48/72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: 71491 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal: 71516 Male

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one hour after treatment.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 24 Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 48 Hour observation.
Both treated eyes appeared normal at the 72-Hour observation.

Other effects:

Bodyweight
Individual bodyweights and bodyweight changes are given in Table 3.
Both animals showed expected gain in bodyweight during the study.

Interpretation of Results

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae         =        (A + B + C) x 2
Score for iris                         =         D x 5
Score for cornea                  =        (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J H and Calandra J C (1962), J. Soc. Cosmet. Chem.13, 281‑289 (seeAppendix3) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.

If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

The results were also interpreted according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Table 1              Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	71491 Male				71516 Male
	IPR= 3				IPR= 0+
Time After Treatment	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
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	1	0	0	0	0	0	0	0
Score (D x 5)	5	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	2	1	0	2	1	1	0
B = Chemosis	2	1	0	0	1	1	0	0
C = Discharge	2	1	0	0	1	0	0	0
Score (A + B + C) x 2	12	8	2	0	8	4	2	0
Total Score	17	8	2	0	8	4	2	0

IPR=  Initial pain reaction

+=     One drop of local anaesthetic instilled into both eyes 1 to 2 minutes before treatment

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
71491 Male	17	8	2	0
71516 Male	8	4	2	0
Group Total	25	12	4	0
Group Mean Score	12.5	6.0	2.0	0.0

Table 3              Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
71491 Male	2.57	2.68	0.11
71516 Male	2.86	2.96	0.10

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulations (EC) No 1272/2008

Conclusions:

The test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008 (EU CLP).
This study is considered to be scientifically justified for use as a key study under Regulation (EC) No. 1907/2006 and the results are appropriate for the purposes of classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

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 to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24 April 2002)

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 two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation.

Conclusion. The test item produced a maximum group mean score of 12.5 and was classified as a mild irritant (Class4 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 Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

A reliable in vitro skin corrosion study according to OECD 431 and in compliance with GLP is available with the test substance (Harlan, 2012). The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKINTM in vitro Reconstructed Human Epidermis (RHE) Model. Duplicate tissues were treated with the test substance for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test substance was rinsed from each tissue before each tissue was taken for MTT-loading. 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.  The optical density (OD) was measured at 540 nm (OD540). The relative mean viability of the test item treated tissues was: 87.1% after 240 minutes exposure, 96.4% after 60 minutes exposure and 110.8% after 3 minutes exposure. The quality criteria required for acceptance of results in the test were satisfied. Thus, the test substance was considered to be Non-Corrosive to the skin.

A reliable in vitro skin irritation study according to OECD 439 and in compliance with GLP is availabe with the test substance (Harlan, 2012). The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model. Triplicate tissues were treated with the test substance 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. 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 the optical density was measured at 540 nm. The relative mean viability of the test item treated tissues was 104.1% after the 15 -minutes exposure period. The quality criteria required for acceptance of results in the test were satisfied. The test item was considered to be Non-Irritant (NI). 

In conclusion, based on the available in vitro skin irritation and corrosion studies the substance is neither corrosive nor irritating.

This results is supported by an in vivo test which was performed prior to test guidelines (Bullock, 1971). The test substance was finely ground, moistened with water and applied at doses of 0.5 g to the skin of New Zealand albino rabbits (2 intact, 2 abraded sites). The exposure period was 24 h and the animals were observed for signs of irritation over a period of 72 h. No signs of skin irritation were observed during the observation period.

 

Eye Irritation:

 A non-reliable in vitro eye irritation study similar to OECD 492 and in compliance with GLP is available with the test substance (Harlan, 2012). The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model. However, at the time of the study no guideline was available. Therefore, there are significant deficiencies from the current test guideline. For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes instead of 4 hours according to test guideline. Triplicate tissues treated with 30 µL of Solution A served as the negative control and triplicate tissues treated with 30 µL of 2% w/v Sodium Dodecyl Sulphate 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. No post-exposure period followed which is also contrary to the current test guideline. 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 relative mean viability of the test material treated tissues after a 10 minute exposure was 107.7%. It was considered unnecessary to proceed with tissue histopathology. The quality criteria required for acceptance of results in the test were satisfied. According to the study plan followed the test item was considered to be a Non-Irritant (NI). However, this study cannot be used for classification as significant deficiencies from the current test guideline are present (reduced exposure time, no post-exposure time).

A reliable in vivo eye irritation study according to OECD 405 and in compliance with GLP is available with the test substance (Harlan, 2012). Two New Zealand White rabbits were treated with approx. 94 mg test substance. The mean score (24/48/72 h) for corneal opacity and iris was 0 for both animals. The mean score (24/48/72 h) for conjunctivae redness was 1 and 0.66, respectively. The means score for conjunctivae chemosis was 0.33 for both animals. The conjunctivae redness was fully reversed after 72 hours and the chemosis after 48 hours.

Therefore, the test substance is considered to be not eye irritating.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

There are no data (study or workplace) to support a classification for respiratory irritation.