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Description of key information

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

In the first in vitro eye irritation study, conducted according to OECD Test Guideline 437 and in compliance with GLP, no conclusion on the eye irritation potential of Cyrene™ could be made (Harlan Cytotest Cell Research GmbH, 2014b).

In the second in vitro eye irritation study, conducted according to the Ocular Irritection Test Method, Cyrene™ was concluded to be irritating to eyes based on IDE score of 13.50 when tested at 125 µL (Eurofins, 2018c).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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 Cytotest Cell Research GmbH, 2014a) 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 by Harlan Cytotest Cell Research GmbH testing facility to perform an in vivo study to obtain a reliable classification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted 24 April 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
MHRA, UK GLP Monitoring Authority
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system 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 following 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 the in vivo skin irritation study, conducted according to OECD Test Guideline 404 and in compliance with GLP, the test material was concluded to be not irritating to skin.
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:
weight of evidence
Study period:
1 Oct. 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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. QA statement)
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
Duration of post- treatment incubation (in vitro):
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.
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
Other effects / acceptance of results:
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). 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 Test Guideline 437 no conclusion can be made on the eye irritation potential of the test substance Cyrene™.

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:
In the in vitro eye irritation study, conducted according to OECD Test Guideline 437 and in compliance with GLP, no conclusion on the eye irritation potential of Cyrene™ can be made.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Date of test: 11 April 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The test method used is not yet a validated OECD test guideline, and the reporting is not up to GLP/OECD TG standards
Justification for type of information:
Irritection Assay to predict potential to cause irritation.
Qualifier:
according to guideline
Guideline:
other: Ocular Irritection Test Method
Deviations:
no
Principles of method if other than guideline:
The proprietary Ocular and Dermal Irritection assays are standardized and quantitative in vitro acute ocular and dermal irritation tests which utilize changes of relevant macromolecules to predict acute ocular and dermal irritancy of chemicals and chemical formulations. Test samples that produce IDE (Irritation Draize Equivalent) of less than or equal to 12.5 are to be considered UK GHS/EU CLP non-irritants. Test samples that produce IDE score greater than 12.5 are to be classified as UN GHS/EU CLP irritants. Test samples that produce IDE score greater than 30.0 are likely to be considered UK GHS/EU CLP Category 1.
GLP compliance:
not specified
Remarks:
No information on GLP compliance has been given in the study report.
Species:
other: Synthetic biobarrier composed of a semi-permeable membrane
Strain:
other: N/A
Amount / concentration applied:
50, 75, 100 and 125 µL
Details on study design:
The Ocular Irritection assay is a quantitative in vitro test method that mimics an acute ocular irritation test. To perform this standardized assay, the test sample is either applied to a synthetic biobarrier composed of a semi-permeable membrane or applied directly to the reagent solution. Following application, the sample is absorbed by and permeates through this synthetic biobarrier to gradually come into contact with a proprietary solution containing glycoproteins. Reaction of the test sample with these proteins and macromolecular complexes promotes conformational changes that may be readily detected as an increase in the turbidity of the protein solution. The ocular irritancy potential of a test sample is expressed as an Irritection Draize Equivalent (IDE) score. This score is defined by comparing the increase in optical density (OD405) produced by the test material to a standard curve that is constructed by measuring the increase in OD produced by a set of Calibration substances.
These Calibrators have been selected for use in these tests because their irritancy potential has been previously documented in a series of in vivo investigations. Test samples producing an IDE score of less than or equal to 12.5 are to be considered not irritating to eyes. Test samples that produce IDE score greater than 12.5 are to be considered eye irritating.
Irritation parameter:
other: IDE score
Run / experiment:
50 µL
Value:
11.4
Remarks on result:
no indication of irritation
Irritation parameter:
other: IDE score
Run / experiment:
75 µL
Value:
12.4
Remarks on result:
no indication of irritation
Irritation parameter:
other: IDE score
Run / experiment:
100 µL
Value:
12.5
Remarks on result:
positive indication of irritation
Irritation parameter:
other: IDE score
Run / experiment:
125 µL
Value:
13.5
Remarks on result:
positive indication of irritation
Interpretation of results:
Category 2B (mildly irritating to eyes) based on GHS criteria
Remarks:
Eye Irritant Category 2 according to CLP Regulation (EC) No 1272/2008.
Conclusions:
In the in vitro eye irritation study, conducted according to the Ocular Irritection Test Method, Cyrene™ was concluded to be irritating to eyes based on IDE score of 13.50 when tested at concentration of 125 µL.
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 Test Guideline 431 and in compliance with GLP, Cyrene™ was concluded to be corrosive as a worst case scenario (Harlan Cytotest Cell Research GmbH, 2014a).

Independent duplicate tissues of the human skin model epiCS® were exposed to 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-minute 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 considered to be 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 by Harlan Cytotest Cell Research GmbH testing facility to perform an in vivo study to obtain a reliable classification.

In the key in vivo skin irritation study, conducted according to OECD Test Guideline 404 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 minute, 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 Laboratories Ltd., 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, 2018b).

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 first in vitro eye irritation study, conducted according to OECD Test Guideline 437 and in compliance with GLP, the test substance, no conclusion on the eye irritation potential of Cyrene™ could be made even though there were no corrosive/severe irritant properties detected. 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) resulting in inconclusive prediction on the eye irritation potential of the substance. No increase of opacity or permeability of the corneas were observed for the negative control. The measured IVIS value of 1.10 lies within the historical range of the IVIS for negative control (0.00 - 2.84). The positive control showed clear opacity and distinctive permeability of the corneas with mean IVIS score of 68.22 corresponding eye damage. Relative to the negative control, the test item caused an increase of the corneal opacity and the permeability (Harlan Cytotest Cell Research GmbH, 2014b).

In the second in vitro eye irritation study, conducted according to the Ocular Irritection Test Method, Cyrene™ was concluded to be irritating to eyes. In the study Cyrene™ was evaluated 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™ is irritating to eyes based on an IDE score of 13.50 when tested at concentration of 125 µL (Eurofins, 2018c).

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 for eye irritation Category 2 "H319: Causes serious eye irritation" is required according to Regulation (EC) No 1272/2008.