Registration Dossier

Administrative data

Description of key information

No skin irritation potential was detected in an in vitro assay (EPISKIN)

An in vitro study for eye irritation potential (BCOP) gave equivocal results. A subsequent in vio eye irritation study gave clear negative results (not irritant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 439 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UN GHS (2003, last rev. 2009)
Deviations:
no
Principles of method if other than guideline:
Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.
GLP compliance:
yes
Species:
other: reconstituted human epidermis model
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Amount / concentration applied:
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.
Duration of treatment / exposure:
60 minutes
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative and the positive control for 60 minutes.
Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following 69.75 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.
Irritation / corrosion parameter:
% tissue viability
Remarks:
expressed as mean relative absorbance
Run / experiment:
all 3
Value:
> 97.8 - < 105.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Results after treatment with the test item and the controls

 

Dose Group

Ex-po-sure Interval

Tissue No.

Absorbance 570 nm
Well 1

Absorbance 570 nm
Well 2

Absorbance 570 nm
Well 3

Mean Absorbance of 3 Wells

Mean-Absorbance

of three wells blank

corrected

Mean

Absorbance

of 3 wells

after blank correction*

Rel. Absorbance [%] Tissue 1, 2 + 3*

Relative Standard Deviation

[%]

Mean Rel. Absorbance

[% of Negative Control]**

Mean Absorbance

Blank corrected viable tissue

without MTT

(Step 2)

Mean Rel. Absorbance

[% of Negative Control]

after colour interference and MTT reduction correction

Blank

 

 

0.036

0.037

0.036

0.036

0.000

 

 

 

 

 

 

Negative Control

60 min

Tissue 1

1.736

1.703

1.695

1.711

1.675

1.673

100.1

1.1

100.0

 

 

Tissue 2

1.679

1.688

1.699

1.689

1.653

98.8

Tissue 3

1.725

1.713

1.740

1.726

1.690

101.0

Positive Control

60 min

Tissue 1

0.116

0.116

0.118

0.117

0.081

0.075

4.8

6.6

4.5

 

 

Tissue 2

0.107

0.111

0.109

0.109

0.073

4.4

Tissue 3

0.108

0.105

0.110

0.107

0.071

4.3

Test Item

60 min

Tissue 1

1.800

1.774

1.835

1.8037

1.767

1.711

105.7

4.0

102.3

0.009

101.8

Tissue 2

1.677

1.685

1.654

1.672

1.636

97.8

Tissue 3

1.765

1.758

1.775

1.766

1.730

103.4

 

* relative absorbance per tissue [rounded values]: 100 x (absorbancetissue) divided by mean absorbance negative control

** relative absorbance per treatment group [rounded values]: 100 x (mean absorbance test item/positive control) divided by (mean absorbance negative control)


Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item passed the MTT interference pre-test. Due to its intensive colour, an additional test with one viable tissue (without MTT addition) was necessary to correct the result in the main experiment.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.5% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 7% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was not reduced (101.8%) after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.49 or 3.24 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.
Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes.
Amount / concentration applied:
0.1 mL of the test item, which was found to weigh approximately 82 mg
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
2
Details on study design:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item, which was found to weigh approximately 82 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Annex 1.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
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) given in Annex 2.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
An additional observation was made 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.
Irritation parameter:
cornea opacity score
Basis:
animal: 75328 Female
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Irritation parameter:
cornea opacity score
Basis:
animal: 75370 Male
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Irritation parameter:
iris score
Basis:
animal: 75328 Female
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
iris score
Basis:
animal: 75370 Male
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
0
Max. score:
2
Reversibility:
other: No effects observed
Irritation parameter:
other: redness
Basis:
animal: 75328 Female
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
other: redness
Basis:
animal: 75370 Male
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal: 75328 Female
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal: 75370 Male
Time point:
other: Highest score at 24, 48, 72 hours and 7 days
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Individual and mean scores for ocular irritation are given in Appendix 1.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye 1 hour after treatment.
Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 48 Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 72 Hour observation.
Both treated eyes appeared normal at the 7 Day observation.
No corrosive effects were noted during the study. The test item did not induce significant or irreversible damage to the rabbit eye.
Other effects:
Body Weight
Individual body weights and body weight change are given in Appendix 2.
Both animals showed expected gain in body weight during the study.

Appendix 1     Eye Irritation Scores

Individual Scores for Eye Irritation

Rabbit Number and Sex

IPR

Evaluation interval

Corneal Opacity

Area of Corneal Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

75328Female

0

1 Hour

0

0

1

2

1

2

75370Male

0

0

0

0

2

1

2

75328Female

 

24 Hour

0

0

0

2

1

1

75370Male

 

0

0

0

2

1

1

75328Female

 

48 Hour

0

0

0

2

1

1

75370Male

 

0

0

0

2

1

0

75328Female

 

72 Hour

0

0

0

1

0

0

75370Male

 

0

0

0

1

1

0

75328Female

 

7 Days

0

0

0

0

0

0

75370Male

 

0

0

0

0

0

0

IPR=Initial pain reaction

Appendix 1 (continued)       Eye Irritation Scores

Mean Values after 24, 48 and 72 Hours

Rabbit Number
and Sex

Number of available data points

Corneal Opacity

Iris

Conjunctivae

Redness

Chemosis

75328Female

3

0.0

0.0

1.7

0.7

75370Male

3

0.0

0.0

1.7

1.0

Assessment According to Regulation (EC) No. 1272/2008

Evaluated Intervals

Corneal Opacity

Iris

Conjunctivae

Redness

Chemosis

24 Hours

Not classified

Not classified

Not classified

Not classified

48 Hours

72 Hours

 

Appendix 2     Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 7

75328Female

3.24

3.30

0.06

75370Male

2.49

2.52

0.03

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the findings in this study, the test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.
Executive summary:

The primary eye irritation potential of the test item was investigated according to a method compatible with OECD test guideline No. 405 and Method B5. The test item was applied by instillation of 0.1 mL into the right eye of each of two young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48, 72 hours and 7 days after test item instillation. 

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iritis, redness and chemosis of the conjunctivae. The individual mean scores for corneal opacity and iritis was 0.0 for both animals. The individual mean scores for the conjunctivae were 1.7 for reddening for both animals and 0.7 or 1.0 for chemosis. 

The instillation of the test item into the eye resulted in conjunctival irritation. These effects were reversible and were no longer evident 7 days after treatment in both animals (end of the observation period). No abnormal findings were observed in the cornea of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No clinical signs of toxicity were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

According to the findings in this study, the test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

no classification

No skin irritation potential was detected in an in vitro assay (EPISKIN)

An in vitro study for eye irritation potential (BCOP) gave equivocal results. A subsequent in vio eye irritation study gave clear negative results (not irritant)