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Description of key information

In the in vitro skin corrosion experiment, conducted according to an appropriate OECD 431 Test Guideline and in compliance with GLP, Cyrene™ was concluded to be corrosive as a worst case scenario in human skin model epiCS®. The value for the 3 minutes treatment did not exceed the threshold for corrosivity of 50%, but the 1 hour treatment value was narrowly below the threshold of 15%. Therefore, the test item was considered to be corrosive (Harlan, 2014).

In the in vivo skin irritation study, conducted according to OECD 404 Test Guideline and in compliance with GLP, the test substance, Cyrene™, was reported to be not irritating to skin (Harlan, 2014b).

In the key in vitro Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, conducted according to OECD 437 Test Guideline and in compliance with GLP, the test substance, Cyrene™, did not reveal corrosive/severe irritant properties (Harlan, 2014c).

Cyrene™ was evaluated with the Irritection Assay System in order to predict its potential to cause ocular irritation. The results indicated that the Cyrene™ was classified as a mild ocular irritant with an IDE score of 13.50, and this finding lead to a UN GHS/EU CLP classification of Category 2B (mildly irritating to eyes) and a conclusion of Eye Irritant Category 2 according to CLP Regulation (EC) No 1272/2008 (Eurofins, 2018).

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:
14 Oct. - 07 Nov. 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
JUSTIFICATION FOR PERFORMING AN IN VIVO STUDY: The in vitro study report (Harlan 2014) concluded the test item to be corrosive to skin. However, this result is related to the specific physico-chemical properties (aprotic polar solvent forming multiphasic systems with water) of the test substance and false positive results in in vitro tests are expected. Therefore, the result is considered to be borderline (inconclusive) and it was recommended to perform an in vivo study to obtain a reliable classification.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted 24 April 2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
MHRA, UK GLP Monitoring Authority
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.55 or 2.74 kg
- Housing: individually housed in suspended cages
- Diet: 2930C Teklad Global Rabbit diet, Harlan Laboratories UK Ltd., Oxon, UK ; ad libitum
- Water: drinking water, ad libitum
- Acclimation period: at least five days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 to 70
- Air changes (per hr): at least fifteen changes
- Photoperiod (hrs dark / hrs light): 12/12The 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.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied: 0.5 mL- Concentration: 100%
Duration of treatment / exposure:
Initial test: 3 min, 1 h and 4 h
Confirmatory test: 4 h
Observation period:
72 hours. Reading time points: 1, 24, 48 and 72 h
Number of animals:
2, male
Details on study design:
TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- Type of wrap if used: The cotton gauze patch, to which the test substance was applied, was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.
REMOVAL OF TEST SUBSTANCE
- Washing: Any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: One patch was removed at each of three time points.
SCORING SYSTEMDraize classification scheme
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No evidence of skin irritation was observed during this study. At all reading time points all scores were "0". No corrosive effects were noted.
Other effects:
- Other adverse local effects: No evidence of skin irritation was noted in any test animal folllowing 3-minute, 1-hour or 4-hour application.
- Other adverse systemic effects: No adverse changes in body weight gain.
Interpretation of results:
GHS criteria not met
Conclusions:
In a skin irritation study, conducted according to OECD 404 Test Guideline and in compliance with GLP, the test material was concluded to be not irritating to skin.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
25 April to 28 April 2014
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Disregarded based on false positive results.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
other: not applicable
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Human
- Sex: Not specified
- Age at study initiation (in days): Not applicable
- Weight at study initiation: Not applicable
- Diet (e.g. ad libitum): Not applicable
- Water (e.g. ad libitum): Not applicable
- Acclimation period: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS kit
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date: 23 April 2014
- Delivery date: not specified
- Date of initiation of testing: 25 April 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% C02
- Temperature of post-treatment incubation (if applicable): not specified

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper.
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: a final concentration of 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter: without reference filter
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: OD570

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability meets the acceptance criterion if the mean OD570 of the two tissues in both treatment intervals are >=0.8.
- Barrier function: not specified
- Morphology: not specified
- Contamination: not specified
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: Duplicate cultures

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 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%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted deionised water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0 N Potassium Hydroxide
Duration of treatment / exposure:
3 minute or 1 hour
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
Duplicate cultures
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
ca. 98.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
ca. 14.2
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: The optical evaluation of the MTT -reducing capacity of the test item after one hour incubation with MTT -reagent did not show evidence of blue colour and thereby was not considered to be an MTT reducer.
- 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
- Range of historical values if different from the ones specified in the test guideline: No
Interpretation of results:
study cannot be used for classification
Conclusions:
In the in vitro skin corrosion experiment, conducted according to an appropriate OECD 431 Test Guideline and in compliance with GLP, Cyrene™ was concluded to be corrosive as a worst case scenario. The value for the 3 minutes treatment did not exceed the threshold for corrosivity of 50%, but the 1 hour treatment value was narrowly below the threshold of 15%. Therefore, the test item was considered to be corrosive under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 Oct. 2014
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
other: cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST METHOD: The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.
IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Schlachthof Bensheim, Bensheim, Germany- Donor animals: at least 9 month old donor cattle
- Date and time of eye collection: 18. Sep. 2014
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) at ambient temperature supplemented with penicillin/streptomycin
PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (vascularization, pigmentation, scratches, opacity): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: cornea holder according to the description given in OEDC guideline 437
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: Minimum Essential Medium (MEM) with sodium bicarbonate and L-glutamine, supplemented immediately before use with 1% [v/v] fetal calf serum- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopical defects, each cornea with an basal opacity > 7 was discarded.
DETERMINATION OF THE BASAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: OP_KiT opacitometer (Electro Design, 63-Riom France)
Vehicle:
unchanged (no vehicle)
Controls:
other: number of corneas for the negative control: 3; number of corneae for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied in the test: 750 µL
POSITIVE CONTROL
- Substance: 2-Ethoxyethanol- Amount(s) applied in the test: 750 µL
NEGATIVE CONTROL
- Substance: (0.9% (w/v) NaCl solution (Saline)- Amount(s) applied in the test: 750 µ
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Number of animals or in vitro replicates:
Number of corneas for the test item: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: The anterior compartment received the test item or negative or positive control at a volume of 750 µL on the surface of the corneas. The corneas were incubated in a horizontal position at 32 ± 1 °C in a water-bath. The incubation time lasted 10 min.
POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance were rinsed off from the anterior compartment with saline. The corneas were incubated at 32 ± 1 °C for further two hours in a vertical position.
DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer
- Time of determination: After further incubation of the corneae in medium for two hours at 32 ± 1 °C in a water-bath, the opacity value was determined (t130).
- Specification of the device: OP_KiT opacitometer (Electro Design, 63-Riom France)
DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
- Amount and concentration of the dye: 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS.
- Incubation time: 90 min at 32 ± 1 °C
Irritation parameter:
cornea opacity score
Run / experiment:
mean / out of 3 corneas / 10 min
Value:
29.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: permeability
Run / experiment:
mean / out of 3 corneas / 10 min
Value:
0.469
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
mean / out of 3 corneas / 10 min
Value:
36.37
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritant / corrosive response data:
With the negative control neither an increase of opacity nor permeability of the corneas could be observed. The measured IVIS value of 1.10 lies within the historical range of the IVIS negative control (0.00 - 2.84).The positive control showed clear opacity and distinctive permeability of the corneas (mean IVIS = 68.22) corresponding to a classification as seriously eye damaging (CLP/EPA/GHS (Cat 1)).Relative to the negative control, the test item caused an increase of the corneal opacity and the permeability. The calculated mean IVIS was 36.37 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Results after 10 Minutes Incubation Time:


Test Group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0.33

0.051

0.051

0.77

1.10

Not categorized

1

0.053

1.80

0

0.049

0.74

Positive Control

53.676

 

0.718*

 

64.44*

68.22

Category 1

61.67

0.906*

75.26*

54.67

0.687*

64.97*

Test Substance

27.67

 

0.582*

 

36.40*

36.37

No prediction can be made

29.67

0.365*

35.14*

30.67

0.461*

37.58*

  *corrected values

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the results of the conducted study, the test item did not reveal corrosive/severe irritant properties. However, according to the in vitro classification criteria defined in OECD 437, no prediction can be made for the test result in regard to eye hazard potency. Therefore, no definitive conclusion for the eye hazardous potential of Cyrene™.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the in vitro skin corrosion experiment, conducted according to an appropriate OECD 431 Test Guideline and in compliance with GLP, Cyrene™ was concluded to be corrosive as a worst case scenario (Harlan, 2014).

Independent duplicate tissues of the human skin model epiCS® were exposed to each 50 µL of the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. Compared to the value of the negative control, after exposure of the test item to the tissues the relative absorbance value decreased to 98.3% after 3 minutes. After the 1 hour exposure the relative absorbance value was reduced to 14.2%. The 3 minutes treatment value did not affect the thresholds for corrosivity (50%), but the value for the 1 hour treatment was narrowly below the threshold of 15%. The study report concluded the test item to be corrosive to skin. However, this result is related to the specific physico-chemical properties (aprotic polar solvent forming multiphasic systems with water) of the test substance and false positive results in in vitro tests are expected. Indeed, the result is borderline (inconclusive). Therefore, it was recommended to perform an in vivo study to obtain a reliable classification.

In the key skin irritation study, conducted according to OECD 404 Test Guideline and in compliance with GLP, the test substance, Cyrene™, was reported to be not irritating to skin. Following single topical application of 0.5 mL of undiluted test material onto rabbit skin for 1 min, 1 hour or 4 hours under semiocclusive dressing, no erythema or edema were observed in either of the test animals. No systemic toxicity effects were reported (Harlan, 2014b).

In a supporting study, Cyrene™ was evaluated in an in vitro Corrositex Test in order to predict its potential to cause skin corrosion. The results of this study indicated that the sample was compatible with the Corrositex system and was concluded to be non-corrosive (Eurofins, 2018).

Based on the available data, Cyrene™ was concluded to be not irritant or corrosive to skin. No irritation was observed when undiluted test material was applied directly to rabbit skin in vivo and borderline results were observed following in vitro application.

In the key in vitro Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, conducted according to OECD 437 Test Guideline and in compliance with GLP, the test substance, Cyrene™, did not reveal corrosive/severe irritant properties. Following single application of 750 µL of undiluted test material to 3 corneas for 10 min at 32 ± 1 °C and further two hours incubation after test item removal, the calculated mean IVIS was 36.37 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made. With the negative control neither an increase of opacity nor permeability of the corneas could be observed. The measured IVIS value of 1.10 lies within the historical range of the IVIS negative control (0.00 - 2.84). The positive control showed clear opacity and distinctive permeability of the corneas (mean IVIS = 68.22) corresponding to a classification as seriously eye damaging (CLP/EPA/GHS (Cat 1)).Relative to the negative control, the test item caused an increase of the corneal opacity and the permeability (Harlan, 2014c).

Cyrene™ was evaluated with the Irritection Assay System in order to predict its potential to cause ocular irritation. The concentrations of sample applied to the reagent solution for analysis were: 50, 75, 100 and 125 µL. The results indicated that the Cyrene™ should be classified as a mild ocular irritant with an IDE score of 13.50, and this finding lead to a UN GHS/EU CLP classification of Category 2B (mildly irritating to eyes) and a conclusion of Eye Irritant Category 2 according to CLP Regulation (EC) No 1272/2008) (Eurofins, 2018).

Justification for classification or non-classification

Based on the available data for Cyrene™, no classification for skin irritation is required according to Regulation (EC) No 1272/2008. However, classification of Category 2 "H319: Causes serious eye damage" is required for eyes according to Regulation (EC) No 1272/2008.