Registration Dossier

Administrative data

Description of key information

Skin corrosion - in vitro, OECD Guideline 431: not corrosive

Skin irritation - in vivo, OECD Guideline 404, two rabbits: not irritating

Eye irritation / corrosion - in vitro, OECD 437: not irritating / not corrosive

Eye irritation - in vivo, OECD 405: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-23 - 2016-08-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The contract to conduct an in vivo skin irritation study was signed before the entry into force of the amendments (Commission Regulation (EU) 2016/863 from 31 May 2016) to Annexes VII and VIII of the REACH Regulation.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate issued by "The Department of Health and of the Government of the United Kingdom" (2016-10-28).
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Leicestershire, UK
- Age at study initiation: 12 to 52 weeks
- Weight at study initiation: 3.5 or 3.66 kg
- Housing: Individually housed in suspended cages. 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 of integrity of the study.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): at least 15 per hr
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Remarks:
with veterinary clippers
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration: used as provided
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
Two females
Details on study design:
TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- Type of wrap if used: cotton gauze patch secured with surgical adhesive tape. The trunk of each rabbit was wrapped in elasticated corset to prevent the animals interfering with the patches.

REMOVAL OF TEST SUBSTANCE
- Washing: Residual test item removed by gentle swabbing with cotton wool soaked in 74% industrial methylated spirits.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later. Test sites were examined for evidence of primary irritation and scored. Any other skin reaction and clinical signs of toxicity, if present, were also recorded.
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Reversibility:
fully reversible within: 24 hours
Irritant / corrosive response data:
Very slight erythema was noted for both treated skin sites at the 24 hour observation. Very slight edema was noted at one treated skin site at the 24 hour observation.
Other effects:
There were no deaths during the study.
Animals showed expected gain in body weight during the study.

Table 1: Individual Skin Reactions.

Skin Reaction Observation Time (following patch removal) Individual Scores Mean 24, 48 and 72 Hour Scores
Rabbit Number and Sex
75517 Female 75518 Female
Erythema/ Eschar Formation Immediately 0 0 0.3
1 Hour 0 0
24 Hours 1 1
48 Hours 0 0
72 Hours 0 0
Edema Formation Immediately 0 0 0.0 and 0.3, respectively
1 Hour 0 0
24 Hours 0 1
48 Hours 0 0
72 Hours 0 0
Interpretation of results:
other: EU GHS (CLP) criteria not met
Remarks:
not irritating
Conclusions:
The test item produced individual mean scores of 0.3 for erythema (both animals) and 0.0 and 0.3 respectively for edema.
Executive summary:

The in vivo study was performed to assess the irritancy of the test item to the skin of New Zealand White rabbit in accordance with OECD Guideline 404 and EU Method B.4 and in compliance with GLP criteria.

A suitable test site was selected at the back of each rabbit. A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured with a strip of surgical adhesive tape. Residual test item removed by gentle swabbing with cotton wool soaked in 74% industrial methylated spirits after a 4 hour exposure of the test item. Observations were made immediatly following removal of the patches and approximately 1, 24, 48 and 72 hours later. The single 4 -hour, semi occluded application of the test item to the intact skin of two rabbits produced very slight erythema at both treated skin sites at the 24 hour observation. Very slight edema was noted at one treated skin site at the 24 hour observation. Mean scores following grating at 24, 48 and 72 hours following patch removal were calculated for erythema and edema. Calculation of the Primary Irritation Index and Grading of Irritancy Potential Using the Daize Scheme. If irreversible alterations of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is considered to be corrosive to rabbit skin. Grading according to Draize may, therefore, not be applicable.

The test item produced individual mean scores of 0.3 for erythema (both animals) and 0.0 and 0.3 respectively for edema.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-29 - 2016-07-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: Method B.40bis (EU Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certicate issued by "The Department of Health of the Government of the United Kingdom".
Test system:
human skin model
Source species:
human
Cell type:
other: epithelial
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit, MatTek
- Tissue batch number(s): EpiDerm™ Tissues (0.63cm2) lot number : 23343
- Delivery date: 28 June 2016
- Assay Medium lot number : 060316ZSA
Upon receipt of the Epiderm™ tissues, the sealed 24-well plate was stored in a refrigerator until use.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently
remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- 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: A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to usage.
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001microplate reader.
- Wavelength: 562nm (OD562)

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is 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
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): as supplied, 100% purity

VEHICLE
- Amount(s) applied (volume or weight with unit): pre-praparation of test material in mineral oil (see test material)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3 minutes and 60 minutes.
Number of replicates:
Two replicates of the test item, the negative control and the positive control for each of the exposure tests (3 minutes and 60 minutes).
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
94.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
95.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- 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.
- Colour interference with MTT: The solution containing the test item was a brown color. This color was considered not to have the potential to cause color interference

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 2.007 for the 3-Minute exposure period and 1.906 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.7% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

The relative mean viability of the test item treated tissues was 94.8% and 95.8% for the 3 minute and 60 minute exposures respectively.

Interpretation of results:
other: EU GHS (CLP) criteria not met
Remarks:
non corrosive
Conclusions:
The relative mean viability of the test item treated tissues was 94.8% and 95.8% for the 3 minute and 60 minute exposures respectively. According to criteria given by Regulation (EC) No 1272/2008 (CLP Regulation) the test item is classified as non-corrosive.
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes in accordance with OECD Guideline 431 and EU Method B.40bis and in compliance with GLP criteria. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

The target cells are epithelial, derived from human skin, and formed into a stratified, comified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.

The quality criteria are fulfilled: The acceptance criteria for negative and positive controlls are fulfilled and the coefficient of variation did not exceed 30%.

The relative mean viability of the test item treated tissues was 94.8% and 95.8% for the 3 minute and 60 minute exposures respectively. According to criteria given by Regulation (EC) No 1272/2008 (CLP Regulation) the test item is classified as non-corrosive.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate issued by "The Department of Health of the Government of the United Kingdom".
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: adult cattle
- Characteristics of donor animals (e.g. age, sex, weight): 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After slaughter eyes were excised by an abattoir employee and placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100IU/mL and streptomycin at 100 µg/mL). Transported to test facility over ice packs on the day of slaughter. The corneas were prepared immediately on arrival.
- indication of any existing defects or lesions in ocular tissue samples: Before and after dissection, all eyes were macroscopically examined. Only corneas free of damage were used.
- Indication of any antibiotics used: HBSS supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): used as provided

Duration of treatment / exposure:
The undiluted item applied for 10 minutes.
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of
damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32+/- 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
three replicates for negative controls, positive controls and test samples, each.

NEGATIVE CONTROL USED
Three negative controls were used.

POSITIVE CONTROL USED
Three positive controls were used.

APPLICATION DOSE AND EXPOSURE TIME
Test item was used as provided (undiluted). The exposure time was 10 minutes at 32+/- 1°C.

POST-INCUBATION PERIOD: 90minutes at 32 +/- 1 °C: Evaluation if the permeability of the corneas to sodium fluorescein.

REMOVAL OF TEST SUBSTANCE
- After the exposure period, the corneas were rinsed three times with fresh complete EMEM containing phenol red befor a final rinse with complete EMEM without phenol red. Then, fresh EMEM without phenol red was added to the corneas.

- POST-EXPOSURE INCUBATION:
60 minutes at 32 +/- 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Calculated by subtracting the inital opacity reading from the final opacity reading. Values were then corrected by subtracting the average change in opacity observed for the negative control corneas. Then the mean opacity value of each treatment group was calculated.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a
microtiter plate reader (Anthos 2001) was measured at a wavelenght of 492 nm (OD492).

- Others:
Histopathology: the corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Value:
2.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: permeability measurement (OD)
Value:
0.015
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The negative control gave opacity of <2.9 and permeability <0.103. The negative control acceptance criteria were therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline:

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment Cornea Number Opacity Permeability (OD) In Vitro Irritancy Score
Pre- Treatment Post- Treatment Post Incubation Post-Incubation-Pre- Treatment Corrected Value   Corrected Value
Negative Control 1 3 3 4 1   0.069    
3 3 4 5 2   0.005    
5 3 4 4 1   0.017    
  1.3a   0.030c   1.8
Positive Control 2 2 27 31 29 27.7 1.373 1.343  
4 4 40 37 33 31.7 1.204 1.174  
6 3 28 31 28 26.7 1.198 1.168  
  28.7b   1.228b 47.1
Test Item 7 3 3 8 5 3.7 0.076 0.046  
8 4 6 6 2 0.7 0.017 0.000  
9 5 5 9 4 2.7 0.018 0.000  
  2.3b   0.015b 2.6

a = mean of the post-incubation – pre-incubation values

b = mean corrected value

c = mean permeability

Interpretation of results:
other: EU GHS (CLP) criteria not met
Remarks:
not classified
Conclusions:
The test item score of 2.6 for the in vitro irritancy score was more than the negative control and less than the positive control.
Executive summary:

The purpose of this in vitro test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage in accordance with OECD Guideline 437 and EU Method B.47 and in compliance with GLP criteria. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of <2.9 and permeability <0.103. The negative control acceptance criteria were therefore satisfied. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

The in vitro irritancy scores for corneas exposed to the test item was 2.6. The mean cornea opacity score for corneas exposed to the test item was 2.3. The mean permeability measurement (OD) for corneas exposed to the test item was 0.015.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-19 - 2016-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The contract to conduct an in vivo eye irritation study was signed before the entry into force of the amendments (Commission Regulation (EU) 2016/863 from 31 May 2016) to Annexes VII and VIII of the REACH Regulation.
The decision to perform an in vivo eye irritation test was also based on expert opinion, that the outcome of the BCOP test (Warren, 2017) was not fully clear and further clarification on the classification was warranted.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate issued by "The Department of Health of the Government of the United Kingdom" (2016-10-28).
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Leicestershire, UK
- Age at study initiation: 12 to 52 weeks
- Weight at study initiation: 2.82 or 3.04 kg
- Housing: Animals were individually housed in suspended cages. Animals were provided with environmental enrichment items which were considered not to contain any contaminants of a level that might have affected the prupose or integrity of the study.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration: Used as provided.
Duration of treatment / exposure:
Single exposure
Observation period (in vivo):
Immediately after administration of the test item, an assessment of the inital pain reaction was made. Additionaly, 12 hours later, the treated animals were checked for signs of pain and suffering.
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:
two male animals
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM: Draize Scale for Scoring Ocular Irritation (presented in "Any other information on materials and methods incl. tables").

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophtalmoscope.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
2
Reversibility:
fully reversible
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible
Remarks on result:
probability of weak irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
1
Reversibility:
fully reversible
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in both treater eyes 1 and 24 hours after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 48-hour observation and in one treated eye at the 72-hour observation.
One treated eye appeared normal at the 72-hour observation and the other treated eye appeared normal at the 7-day observation.
Other effects:
There were no deaths or remarkable body weight changes during the study period.

Table 1: Individual and Mean Scores for Cornea, Iris and Conjunctivae.

Rabbit Number and Sex Time After Treatment Corneal Opacity Iridial Inflammation Conjunctival Redness ConjunctivalChemosis
75571 Male 24 Hours 0 0 2 1
48 Hours 0 0 1 1
72 Hours 0 0 1 1
Total 0 0 4 3
Mean 0.0 0.0 1.3 1.0
75561 Male 24 Hours 0 0 2 1
48 Hours 0 0 1 1
72 Hours 0 0 0 0
Total 0 0 3 2
Mean 0.0 0.0 1.0 0.7
Interpretation of results:
other: EU GHS (CLP) criteria not met
Remarks:
not irritating to the eye
Conclusions:
No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 48-Hour observation and in one treated eye at the 72-Hour observation. One treated eye appeared normal at the 72-Hour observation and the other treated eye appeared normal at the 7-Day observation.
Both animals showed a slight expected gain in body weight during the study.
The test item produced individual mean scores of 0.0 for corneal opacity, 0.0 for iritis, 1.3 or 1.0 respectively for conjunctival redness and 1.0 or 0.7 respectively for conjunctival chemosis.
Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit in accordance with OECD Guideline 405 and EU Method B.5 and in compliance with GLP criteria.

A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye of two rabbits in a single application, formed by gently pulling the lower lid away from the eyeball. 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. The treated animal was checked for signs of pain and suffering approximately 12 hours later. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977). Any other ocular effects were also noted. Any clinical signs of toxicity, if present, were also recorded. An additional observation was made in one treated eye on Day 7 to assess the reversibility of the ocular effects. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period. No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 48-Hour observation and in one treated eye at the 72-Hour observation. One treated eye appeared normal at the 72-Hour observation and the other treated eye appeared normal at the 7-Day observation. Both animals showed a slight expected gain in body weight during the study. The test item produced individual mean scores of 0.0 for corneal opacity, 0.0 for iritis, 1.3 or 1.0 respectively for conjunctival redness and 1.0 or 0.7 respectively for conjunctival chemosis.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

The purpose of this in vitro key test (Warren, 2017) is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes in accordance with OECD Guideline 431 and EU Method B.40bis and in compliance with GLP criteria.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

The target cells are epithelial, derived from human skin, and formed into a stratified, comified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.

The quality criteria are fulfilled: The acceptance criteria for negative and positive controls are fulfilled and the coefficient of variation did not exceed 30%.

The relative mean viability of the test item treated tissues was 94.8% and 95.8% for the 3 minute and 60 minute exposures respectively. According to criteria given by Regulation (EC) No 1272/2008 (CLP Regulation) the test item is classified as non-corrosive.

The in vivo key study (Sanders, 2016b) was performed to assess the irritancy of the test item to the skin of New Zealand White rabbit in accordance with OECD Guideline 404 and EU Method B.4 and in compliance with GLP criteria. A suitable test site was selected at the back of each rabbit. A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured with a strip of surgical adhesive tape. Residual test item removed by gentle swabbing with cotton wool soaked in 74% industrial methylated spirits after a 4 hour exposure of the test item. Observations were made immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later. The single 4 -hour, semi occluded application of the test item to the intact skin of two rabbits produced very slight erythema at both treated skin sites at the 24 hour observation. Very slight edema was noted at one treated skin site at the 24 hour observation. Mean scores following grating at 24, 48 and 72 hours following patch removal were calculated for erythema and edema. Calculation of the Primary Irritation Index and Grading of Irritancy Potential using the Daize Scheme. If irreversible alterations of the dermal tissue are noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is considered to be corrosive to rabbit skin. Grading according to Draize may, therefore, not be applicable.

The test item produced individual mean scores of 0.3 for erythema (both animals) and 0.0 and 0.3 respectively for edema.

Eye irritation:

The purpose of this in vitro key study was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage in accordance with OECD Guideline 437 and EU Method B.47 and in compliance with GLP criteria. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of <2.9 and permeability <0.103. The negative control acceptance criteria were therefore satisfied. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

The in vitro irritancy score for corneas exposed to the test item was 2.6. The mean cornea opacity score for corneas exposed to the test item was 2.3. The mean permeability measurement (OD) for corneas exposed to the test item was 0.015.

The in vivo key study (Sanders, 2016c) was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit in accordance with OECD Guideline 405 and EU Method B.5 and in compliance with GLP criteria. This decision was based on expert opinion, that the outcome of the BCOP test (Warren, 2017) was not fully clear and further clarification on the classification was warranted.

A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye of two rabbits in a single application, formed by gently pulling the lower lid away from the eyeball. 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. The treated animal was checked for signs of pain and suffering approximately 12 hours later. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977). Any other ocular effects were also noted. Any clinical signs of toxicity, if present, were also recorded. An additional observation was made in one treated eye on Day 7 to assess the reversibility of the ocular effects. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period. No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 48-Hour observation and in one treated eye at the 72-Hour observation. One treated eye appeared normal at the 72-Hour observation and the other treated eye appeared normal at the 7-Day observation. Both animals showed a slight expected gain in body weight during the study. The test item produced individual mean scores of 0.0 for corneal opacity, 0.0 for iritis, 1.3 or 1.0 respectively for conjunctival redness and 1.0 or 0.7 respectively for conjunctival chemosis.

Justification for classification or non-classification

There are well documented in vitro and in vivo guideline studies available for the magnesium metaborate, determining the dermal irritation/corrosion and the irritating/corrosive potential to the eye.

The results of the in vitro EpiDerm™ Human Skin Model revealed a relative mean viability of the test item treated tissues of 94.8% and 95.8% for the 3 minute and 60 minute exposures respectively. The test item is classified as non corrosive in this in vitro test. In the in vivo test on dermal irritation the test item produced individual mean scores of 0.3 for erythema (both animals) and 0.0 and 0.3 respectively for edema. The test item was not a dermal irritant.

The in vitro irritancy scores in the BCOP assay for corneas exposed to the test item was 2.6. The mean cornea opacity score for corneas exposed to the test item was 2.3. The mean permeability measurement (OD) for corneas exposed to the test item was 0.015. The test item was not corrosive. The in vivo test on eye irritation showed individual mean scores of 0.0 for corneal opacity, 0.0 for iritis, 1.3 or 1.0 respectively for conjunctival redness and 1.0 or 0.7 respectively for conjunctival chemosis for the test item. The test item ist not an eye irritant.

According to these results the test item magnesium metaborate does not fulfil the classification criteria of Regulation (EC) No 1272/2008 (CLP Regulation) for dermal irritation /corrosion and irritation hazard to the eye.