Registration Dossier

Administrative data

Description of key information

Introduction

 The study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit.

 

Results

 A single 4-Hour, semi occluded application of the test item to the intact skin of three rabbits produced very slight to well-defined erythema and very slight to slight edema. Other skin reactions noted were light brown discoloration of the epidermis, loss of skin elasticity, slight desquamation and glossy skin. One treated skin site appeared normal at the 72-Hour observation and one other treated skin site appeared normal at the 7-Day observation. No corrosive effects were noted.

Conclusion

The test item produced individual mean scores of 2.0, 1.0 and 0.7 for erythema and 1.7, 1.0 and 0.3 for edema. In accordance with EU CLP Regulation (EC) No. 1272/2008, classification of this substance is not required for dermal irritation as scores for erythema and oedema did not fulfil the criteria for classification and were fully reversible by the end of the 14-day observation period.[LE1] 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 26 January 2016 Experimental completion date 12 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 28 July 2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
Method B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Remarks:
New Zealand White (Hsdlf:NZW)
Details on test animals and environmental conditions:
Three 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 2.69 or 4.00 kg and were 12 to 20 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.

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.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
A quantity of 0.5 mL of the test item was applied directly to the skin
Duration of treatment / exposure:
Four hours
Observation period:
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation
Number of animals:
3
Details on study design:
Please see below
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
mean
Time point:
24/48/72 h
Score:
1.23
Max. score:
2
Reversibility:
not fully reversible within:
Remarks on result:
other: The test item produced individual mean scores of 2.0, 1.0 and 0.7 for erythema
Remarks:
Very slight erythema, with or without very slight edema, were noted at all treated skin sites immediately after patch removal. Well-defined erythema and slight edema were noted at one treated skin site and very slight erythema and very slight edema were noted at two treated skin sites 1 hour after patch removal. Well-defined erythema and slight edema were noted at one treated skin site and very slight erythema, with or without very or slight edema, were noted at two treated skin sites at the 24 and 48-Hour observations. Well-defined erythema and very slight edema were noted at one treated skin site and very slight erythema was noted at one other treated skin site at the 72-Hour observation. At the 72-Hour observation, light brown discoloration of the epidermis and loss of skin elasticity were also noted at one treated skin site and slight desquamation was noted at one other treated skin site. Slight desquamation and glossy skin were noted at one treated skin site at the 7-Day observation.
Irritant / corrosive response data:
Very slight erythema, with or without very slight edema, were noted at all treated skin sites immediately after patch removal. Well-defined erythema and slight edema were noted at one treated skin site and very slight erythema and very slight edema were noted at two treated skin sites 1 hour after patch removal. Well-defined erythema and slight edema were noted at one treated skin site and very slight erythema, with or without very or slight edema, were noted at two treated skin sites at the 24 and 48-Hour observations. Well-defined erythema and very slight edema were noted at one treated skin site and very slight erythema was noted at one other treated skin site at the 72-Hour observation.

At the 72-Hour observation, light brown discoloration of the epidermis and loss of skin elasticity were also noted at one treated skin site and slight desquamation was noted at one other treated skin site. Slight desquamation and glossy skin were noted at one treated skin site at the 7-Day observation.

Skin Reactions

 

The individual scores and mean scores for erythema/eschar and edema are given in the attached Appendix 1.

 

Very slight erythema, with or without very slight edema, were noted at all treated skin sites immediately after patch removal. Well-defined erythema and slight edema were noted at one treated skin site and very slight erythema and very slight edema were noted at two treated skin sites 1 hour after patch removal. Well-defined erythema and slight edema were noted at one treated skin site and very slight erythema, with or without very or slight edema, were noted at two treated skin sites at the 24 and 48-Hour observations. Well-defined erythema and very slight edema were noted at one treated skin site and very slight erythema was noted at one other treated skin site at the 72-Hour observation.

 

At the 72-Hour observation, light brown discoloration of the epidermis and loss of skin elasticity were also noted at one treated skin site and slight desquamation was noted at one other treated skin site. Slight desquamation and glossy skin were noted at one treated skin site at the 7-Day observation.

 

The third treated skin site appeared normal at the 72-Hour observation and one of the initial treated skin sites appeared normal at the 7-Day observation.

 

BodyWeight

 

Individual body weights and body weight change are given in the attached Appendix 2. All animals showed expected gain in body weight during the study.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item produced individual mean scores of 2.0, 1.0 and 0.7 for erythema and 1.7, 1.0 and 0.3 for edema.
Executive summary:

Introduction

 The study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit.

 Results

A single 4-Hour, semi occluded application of the test item to the intact skin of three rabbits produced very slight to well-defined erythema and very slight to slight edema. Other skin reactions noted were light brown discoloration of the epidermis, loss of skin elasticity, slight desquamation and glossy skin. One treated skin site appeared normal at the 72-Hour observation and one other treated skin site appeared normal at the 7-Day observation. No corrosive effects were noted.

 

Conclusion

 The test item produced individual mean scores of 2.0, 1.0 and 0.7 for erythema and 1.7, 1.0 and 0.3 for edema.

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:
Experimental start date: 11 February 2016 Experimental completion date: 11 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: The Rabbit Enucleated Eye Test
Version / remarks:
The Rabbit Enucleated Eye Test has been included in evaluations of the validity of eye irritancy test methods, but is not currently considered to be a validated 'stand-alone' test method. No formally adopted test guideline is available, but a protocol has been proposed which was based upon the method detailed in this document (ICCVAM, 2006 and 2009).

A positive result in the rabbit enucleated eye test is accepted within the EU as an indication of severe eye irritancy potential without the need for confirmation in a rabbit eye irritation test (European Chemicals Bureau, 2006).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals were 12 to 20 weeks old. Rabbits that have previously been used in skin or eye irritation studies at the test facility may. be used as eye donors. If the donor animals have been used in an eye irritation study, only the untreated eyes were used.
Vehicle:
unchanged (no vehicle)
Remarks:
For the purpose of the study the test item was used as supplied.
Controls:
yes
yes, concurrent no treatment
Amount / concentration applied:
Three eyes were treated with the test item. Two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally into a petri dish.

The test item was used undiluted as supplied.
A volume of 0.1 mL of the test item was applied as evenly as possible to the surface of the cornea
Duration of treatment / exposure:
After ten seconds the test item was washed off the cornea using a minimum of 20 mL of 0.9% saline solution (pre-warmed to approximately 32 °C).

Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye.

The untreated eyes were similarly washed and used for control purposes.
Duration of post- treatment incubation (in vitro):
Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye.
Number of animals or in vitro replicates:
5 eyes
Details on study design:
Please see below
Irritation parameter:
percent corneal swelling
Remarks:
Mean Corneal Swelling
Run / experiment:
60 runs
Value:
31.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean Corneal Swelling
Run / experiment:
120 runs
Value:
54.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean Corneal Swelling
Run / experiment:
240 runs
Value:
90
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Corneal Opacity

Individual scores for corneal opacity are given in the attached Appendix 1.

Some loss of transparency was noted in all test eyes. No corneal effects were noted in the control eyes during the study period.

Corneal Thickness

Individual and mean measurements for corneal thickness are given in the attached Appendix 2. The determination of corneal swelling is given in Table 1 and Table 2. The calculated mean corneal swellings are given in Table 3.

Corneal swelling of the test eyes was considerably greater than that observed in the control eyes over the same period and exceeded the 2.25% cut-off value.

Corneal Condition

The condition of the corneal epithelium following treatment is given in the attached Appendix 3.

Sloughing of the corneal epithelium was noted in test eyes. The condition of the corneal epithelium of the control eyes appeared normal during the study.

Fluorescein Uptake

Individual scores for fluorescein uptake are given in the attached Appendix 4.

Fluorescein uptake was noted in the test eyes 240 minutes following test item application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Table 1            Determination of Corneal Swelling-Test Eyes

 

Chamber Number

 

Observation Period (minutes)

Mean Corneal Thickness(µm)

 

Corneal Swelling(%)a

 

Chamber Number

 

Observation Period (minutes)

Mean Corneal Thickness(µm)

 

Corneal Swelling(%)a

 

Chamber Number

 

Observation Period (minutes)

Mean Corneal Thickness(µm)

Corneal Swelling(%)a

 

 

 

 

 

 

IA

Post equilibration

 

355.8

 

na

 

 

 

 

 

 

3A

Post equilibration

 

400.0

 

na

 

 

 

 

 

 

5A

Post equilibration

 

414.2

 

na

60 Post treatment

 

520.2

 

46.2

60 Post treatment

 

514.0

 

28.5

60 Post treatment

 

493.8

 

19.2

120 Post treatment

 

643.8

 

80.9

120 Post treatment

 

578.4

 

44.6

120 Post treatment

 

573.6

 

38.5

180 Post treatment

 

771.6

 

116.9

180 Post treatment

 

612.8

 

53.2

180 Post treatment

 

633.8

 

53.0

240 Post treatment

 

854.0

 

140.0

240 Post treatment

 

655.4

 

63.9

240 Post treatment

 

688.6

 

66.2

a = % corneal swelling = (mean corneal thickness post-treatment) - (mean corneal thickness post equilibration) / mean corneal thickness post equilibrium x 100

Na = Not applicable

Table2            Determination of Corneal Swelling - Control Eyes

 

ChamberNumber

 

Observation Period (minutes)

 

Mean Corneal Thickness (µm)

 

Corneal Swelling(%)a

 

Chamber Number

 

Observation Period(minutes)

 

Mean Corneal Thickness (µm)

 

Corneal Swelling(%)a

 

 

 

 

 

 

2A

 

Post equilibration

 

353.0

 

na

 

 

 

 

 

 

4A

 

Post equilibration

 

403.8

 

na

 

60 Post treatment

 

368.4

 

4.4

 

60 Post treatment

 

449.8

 

11.4

 

120 Post treatment

 

356.4

 

1.0

 

120 Post treatment

 

472.6

 

17.0

 

180 Post treatment

 

350.4

 

0.0

 

180 Post treatment

 

468.4

 

16.0

 

240 Post treatment

 

348.6

 

0.0

 

240 Post treatment

 

466.6

 

15.6

a = % corneal swelling = (mean corneal thickness post-treatment) - (mean corneal thickness post equilibration) / mean corneal thickness post equilibrium x 100

Na = Not applicable

Table3            Mean Corneal Swelling

 

 

Time After Treatment

 

60 minutes

 

120 minutes

 

240 minutes

 

Test Eyes

 

31.3+

 

54.7+

 

90.0+

 

Control Eyes

 

7.9

 

9.0

 

7.8

+ = Meets or exceeds cut-off value indicating a severe ocular irritant

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Following assessment of the data for all endpoints, the test item was considered to have the potential to cause severe ocular irritancy in viva.
Executive summary:

Introduction

 Itis a legal and ethical duty under the Animals (Scientific Procedure) Act 1986 that, in the interest of animal welfare, the unnecessary use of animals is avoided, and that any testing which is likely to produc esevere responses in animals is minimized.

 Therefore, before in vivo irritation testing is performed, all existing information on the test item, or its analogues should be reviewed. Available information indicated that the test item had the potential to produce severe effects in a rabbit eye and to confirm this initial assessment, a Rabbit Enucleated Eye Test (REET) was performed.

 

This step-wise procedure is in accordance with OECD Test Guideline 405,UK Home Office regulations and Envigo Research Limited Ethical Testing Strategy.

A study was performed to assess the ocular irritancy potential of the test item in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test item in man.

 

Method

 0.1 mL of the test item was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 ±1.5 °C within the superfusion chamber. A further two enucleated eyes remained untreated for control purposes.

Results

 Maximal ocular irritation observations recorded for the test eyes were as follows:

 

Corneal Opacity

FluoresceinUptake

Mean Corneal Swelling(%)

 

Condition of Corneal Epithelium

Test Eyes a

Control Eyes b

Cldy x Area

Int x Area

60

ruins

120

ruins

240

ruins

60

ruins

120

ruins

240

ruins

3

4+

31.3+

54.7+

90.0+

7.9

9.0

7.8

sloughing+

 

  

a=          For each time point the swelling recorded is the mean of three eyes

b=         For each time point the swelling recorded is the mean of two eyes

Cldy =   Corneal cloudiness

Int=       Intensity of fluoresceinuptake

mms = Minutes following treatment

+=         Meets or exceeds cut-off value indicating a severe ocular irritant


 

Conclusion

 Following assessment of the data for all endpoints the test item was considered to have the potential to cause severe ocular irritancy in vivo.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Introduction

 It is a legal and ethical duty under the Animals (Scientific Procedure) Act 1986 that, in the interest of animal welfare, the unnecessary use of animals is avoided, and that any testing which is likely to produc e severe responses in animals is minimized.

 Therefore, before in viva irritation testing is performed, all existing information on the test item, or its analogues should be reviewed. Available information indicated that the test item had the potential to produce severe effects in a rabbit eye and to confirm this initial assessment, a Rabbit Enucleated Eye Test (REET) was performed.

 

This step-wise procedure is in accordance with OECD Test Guideline 405,UK Home Office regulations and Envigo Research Limited Ethical Testing Strategy.

 

A study was performed to assess the ocular irritancy potential of the test item in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test item in man.

 

Method

 0.1 mL of the test item was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 ±1.5 °C within the superfusion chamber. A further two enucleated eyes remained untreated for control purposes.

Results

 Maximal ocular irritation observations recorded for the test eyes were as follows:

 

Corneal Opacity

FluoresceinUptake

Mean Corneal Swelling(%)

 

Condition of Corneal Epithelium

Test Eyes a

Control Eyes b

Cldy x Area

Int x Area

60

ruins

120

ruins

240

ruins

60

ruins

120

ruins

240

ruins

3

4+

31.3+

54.7+

90.0+

7.9

9.0

7.8

sloughing+

 

  

a=          For each time point the swelling recorded is the mean of three eyes

b=         For each time point the swelling recorded is the mean of two eyes

Cldy =  Cornealc loudiness

Int=       Intensity of fluoresceinu ptake

mms = Minutes following treatment

+=         Meets or exceeds cut-off value indicating a severe ocular irritant


 

Conclusion

 

Following assessment of the data for all endpoints the test item was considered to have the potential to cause severe ocular irritancy in vivo.

Justification for classification or non-classification

Following assessment of the data for all endpoints the test item was considered to have the potential to cause severe ocular irritancy in vivo.