Registration Dossier

Administrative data

Description of key information

SKIN IRRITATION

In vitro skin corrosion

Under the conditions of the study, the mean cell viability for the test material was 48.3 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive.

In vitro skin irritation

Under the conditions of the study, the mean cell viability of the test material was 100.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

Sax's Handbook

According to the handbook data the substance is a skin irritant.

Food and chemical Toxicology

The substance was applied full strength to intact or abraded rabbit skin for 24 hours under occlusion and was found to be moderately irritating.

Tested at 2 % in petrolatum, the substance produced no irritation after a 48 hour closed-patch test on human subjects.

EYE IRRITATION

Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is classified as severely irritating, UN GHS Classification: Category 1.

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:
supporting study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Valid with restrictions. This is peer reviewed data where the test methodology and identity of the substance have been evaluated, and a reliable and representative value for the endpoint has been selected. No information on GLP status or test guideline followed is available.
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
Handbook data does not specify the method. Data from peer reviewed source.
GLP compliance:
not specified
Test material information:
Composition 1
Species:
rabbit
Strain:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 500 mg
Duration of treatment / exposure:
24 hours
Irritation parameter:
overall irritation score
Remarks on result:
other: a skin irritant
Interpretation of results:
study cannot be used for classification
Conclusions:
According to the handbook data the substance is a skin irritant.
Executive summary:

According to the handbook data the substance is a skin irritant.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
1. The substance was applied full strength to intact or abraded rabbit skin for 24 hours under occlusion was moderately irritating (primary reference: Moreno, O.M. (1977) Report to RIFM, 7 October)
2. The substance was tested at 2% in petrolatum in a 48 hour closed-patch test on human subjects (primary reference: Kilgman, A.M. (1977) Report to RIFM, 6 June)
GLP compliance:
not specified
Test material information:
Composition 1
Species:
other: rabbits and humans
Strain:
not specified
Type of coverage:
occlusive
Preparation of test site:
other: intact or abraded rabbit skin
Vehicle:
unchanged (no vehicle)
Remarks:
unchanged for rabbits, petrolatum for humans
Number of animals:
Human subjects: 25
Irritation parameter:
overall irritation score
Basis:
other: Rabbit
Time point:
24 h
Remarks on result:
other: moderately irritating
Irritation parameter:
overall irritation score
Basis:
other: Human subjects
Time point:
48 h
Remarks on result:
no indication of irritation
Interpretation of results:
study cannot be used for classification
Conclusions:
The substance was applied full strength to intact or abraded rabbit skin for 24 hours under occlusion and was found to be moderately irritating.
Tested at 2 % in petrolatum, the substance produced no irritation after a 48 hour closed-patch test on human subjects.
Executive summary:

The substance was applied full strength to intact or abraded rabbit skin for 24 hours under occlusion and was found to be moderately irritating.

Tested at 2 % in petrolatum, the substance produced no irritation after a 48 hour closed-patch test on human subjects.

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:
15 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
The solubility of the test material in physiological saline was tested prior to the experiment (30 mg test material in 1 mL physiological saline). The test material dissolved in physiological saline.
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to the Test Facility at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the Test Facility and processed within 2 hours of collection.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration (if solution): Benzalkonium chloride solution 5 % (w/v)

NEGATIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration (if solution): physiological saline (0.9 % (w/v) NaCl) solution
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
Three test material treated eyes, three positive control treated eyes and one negative control eye were examined during the study.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes Selection:
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

- Preparation of eyes:
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

- Examination of eyes:
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus.
The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
- Acclimatisation time:

If the selected eyes were appropriate for the test, acclimatisation started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatisation and treatment periods.

- Baseline Assessments
At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes in the experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
: Three test material treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. For treatment, 30 µL of the test material was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 µL of benzalkonium chloride solution 5 % (w/v). The negative control eye was treated with 30 µL of physiological saline (0.9 % (w/v) NaCl) solution.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.

Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.


MEASURED ENDPOINTS:
- Corneal swelling
- Corneal opacity
- Fluorescein retenion

Corneal swelling was calculated according to the following formulae:
CS at time t = ((CT at time t - CT at t=0) /CT at t=0) x 100
Mean CS at time t = (FECS(at time t)+ SECS(at time t) + TECS(at time t)) / 3

CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point
Small negative numbers for swelling (0 to -5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

Cornea opacity was calculated according to the following formulae:
ΔCO at time t = CO at time t – CO at t=0
Mean ΔCOmax = (FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)) / 3

CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the posttreatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:
ΔFR at time t = FR at time t – FR at t=0
Mean ΔFR = (FEFR (30min) + SEFR(30min) + TEFR(30min)) / 3

FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention


DECISION CRITERIA:
> Criteria for “No category” (all true)
- 3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II
- No severe corneal morphological changes
- Test material was not stuck to the cornea at 240 minutes after the post-treatment rinse

> Criteria for “Category 1” (one or more true)
- 2 or more endpoints classed as IV
- Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
- Corneal opacity = 4 at any time point (in at least 2 eyes)
- Severe loosening of epithelium (in at least 1 eye)

> Criteria for “No prediction can be made” (one or two true)
- Based on the endpoints not classifiable for No Category, or for Category 1
- Particles of test material were stuck to the cornea and could not be washed off during the study
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
2.67
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
2.7
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Mean maximum corneal swelling at up to 75 min
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
5.9
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Mean maximum corneal swelling at up to 240 min
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
3
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
TEST MATERIAL
Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is classified as severely irritating, UN GHS Classification: Category 1

POSITIVE CONTROL
The positive control Benzalkonium chloride solution (5 % (w/v) was classified as severely irritating, UN GHS Classification: Category 1.

NEGATIVE CONTROL
The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

VALIDITY OF THE TEST
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid

Table 1: Results

 Treatment     Test Material     Positive control     Negative control
 Observation  Value  ICE Class  Value  ICE Class Value  ICE Class
 Mean maximum corneal swelling at up to 75 min  2.7 %  I  11.5 %  II  0.0 %  I
 Mean maximum corneal swelling at up to 240 min  5.9 %  II  27.5 %  III  0.0 %  I
 Mean maximum corneal opacity  2.67  IV  4.00  IV  0.00  I
 Mean fluorescein retention  3.00  IV  3.00  IV  0.00  I
 Other Observations     None Severe loosening of epithelium was observed in one eye at 120 minutes after the post- treatment rinse  

 None

 
 Overall ICE Class     1 x II, 2 x IV     1 x III, 2 x IV     3 x I
Interpretation of results:
other: EU Criteria: Category 1
Conclusions:
Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is classified as severely irritating, UN GHS Classification: Category 1
Executive summary:

The potential of the substance to be an eye irritant was investigated in vitro following the standardised guideline OECD 438, under GLP conditions.

During the study, after the zero reference measurements, the eyes were held in a horizontal position and the test material was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL test material. The three positive control eyes were treated in a similar way with 30 µL benzalkonium chloride solution 5 % (w/v). The negative control eye was treated with 30 µL of physiological saline (0.9 % (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in the experiment. Thus, the experiment was considered to be valid.

Slight corneal swelling (mean 5.9 %) was observed during the four-hour observation period on test material treated eyes. Severe cornea opacity change (severity 2 on one eye and severity 3 on two eyes) was observed on three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is classified as severely irritating, UN GHS Classification: Category 1.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION

In vitro skin corrosion

The corrosivity of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 431 in a reconstructed human epidermis model, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, disks of EPISKIN™ (SM) were treated with the test material and incubated for 4 hours at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control). For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is < 35 % the test material is considered to be corrosive to skin.

Under the conditions of the study, the mean cell viability for the test material was 48.3 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive.

In vitro skin irritation

The skin irritation potential of the test material was investigated in vitro in accordance with the standardised guidelines OECD 439 and EU Method B.46 in a reconstructed human epidermis model, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, disks of EPISKIN™ (SM) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2, in a > 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS treated epidermis were used as negative control and Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test material is considered to be irritant to skin.

Following exposure with the test material the mean cell viability was 100.0% compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

Sax's Handbook

According to the handbook data the substance is a skin irritant.

Food and chemical Toxicology

The substance was applied full strength to intact or abraded rabbit skin for 24 hours under occlusion was moderately irritating.

Tested at 2 % in petrolatum, the substance produced no irritation after a 48 hour closed-patch test on human subjects.

EYE IRRITATION

The potential of the substance to be an eye irritant was investigated in vitro following the standardised guideline OECD 438, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, after the zero reference measurements, the eyes were held in a horizontal position and the test material was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL test material. The three positive control eyes were treated in a similar way with 30 µL benzalkonium chloride solution 5 % (w/v). The negative control eye was treated with 30 µL of physiological saline (0.9 % (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in the experiment. Thus, the experiment was considered to be valid.

Slight corneal swelling (mean 5.9 %) was observed during the four-hour observation period on test material treated eyes. Severe cornea opacity change (severity 2 on one eye and severity 3 on two eyes) was observed on three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is classified as severely irritating, UN GHS Classification: Category 1

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to skin corrosion/ irritation but does require classification for irreversible effects on the eye (Category 1).