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Description of key information

Skin irritation
In an in vitro study, duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The relative mean viabilities for each test item treated group were 95.6 and 100.1% at the end of 3 and 60 minutes exposure period. Hence, the test item was considered to be non-corrosive to the skin.


Additionally, an in vivo study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit. A single 4-Hour, semi-occluded application of the test item to the intact skin of two rabbits produced well-defined erythema, slight edema and loss of skin elasticity and flexibility. Both treated skin sites appeared normal at the 7-Day observation. The test item produced a primary irritation index of¨3.8 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.

No corrosive effects were noted.

However, the test item was classified as non irritant according to the Globally Harmonized System of Classification and Labelling of Chemicals, since the effects found were fully reversible by the end of 7 days and mean scores for erythema and edema were <2.3.

Eye irritation
In a bovine corneal opacity study, the test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS calculated for test item group was 5.5. No prediction of eye irritation can be made.

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:
The study was conducted between 14 April 2016 and 21 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Remarks:
Two male rabbits
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Leicestershire, UK.
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.53 or 2.73 kg
- Housing :individually housed in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

IN-LIFE DATES: From: 14 April 2016 To: 21 April 2016
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
2
Details on study design:
TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- Type of wrap if used: To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): gentle swabbing with cotton wool soaked in 74% industrial methylated spirits
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1, 24, 48 and 72 hours

SCORING SYSTEM:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation
- No erythema : 0
- Very slight erythema (barely perceptible): 1
- Well-defined erythema: 2
- Moderate to severe erythema: 3
- Severe erythema (beef redness) to eschar formation preventing grading of erythema: 4

Edema Formation
- No edema: 0
- Very slight edema (barely perceptible): 1
- Slight edema (edges of area well-defined by definite raising): 2
- Moderate edema (raised approximately 1 millimeter): 3
- Severe edema (raised more than 1 millimeter and extending beyond the area of exposure): 4

Any other skin reactions and clinical signs of toxicity, if present, were also recorded.
An additional observation was made on Day 7 to assess the reversibility of skin reactions.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

DATA EVALUATION
The scores for erythema and edema at the 24 and 72 Hour readings were totaled for the two test rabbits (8 values) and this total was divided by 4 to give the primary irritation index of the test item. The test item was classified according to the following scheme devised by Draize, J.H. (1959):

- Method of calculation: Draize, J.H. (1959)
Primary Irritation Index Classification of Irritancy
0 Non-irritant
> 0 to 2 Mild irritant
> 2 to 5 Moderate irritant
> 5 to 8 Severe irritant

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.
The results were also interpreted according to the Globally Harmonized System of Classification and Labelling of Chemicals.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: Immediately
Score:
0
Remarks on result:
other: Orange colored staining of the test site
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: 1 hour
Score:
0
Remarks on result:
other: Orange colored staining of the test site
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Orange colored staining of the test site
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
48 h
Score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Orange colored staining of the test site, Loss of skin elasticity
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
72 h
Score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Orange colored staining of the test site, Loss of skin elasticity, Loss of skin flexibility
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
7 d
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: Immediately
Score:
0
Remarks on result:
other: Orange colored staining of the test site
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: 1 hour
Score:
0
Remarks on result:
other: Orange colored staining of the test site
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24 h
Score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Orange colored staining of the test site
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
48 h
Score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Orange colored staining of the test site, Loss of skin elasticity
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
72 h
Score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Orange colored staining of the test site, Loss of skin elasticity, Loss of skin flexibility
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
7 d
Score:
0
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: Immediately
Score:
0
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 1 hour
Score:
0
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24 h
Score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
48 h
Score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
72 h
Score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #1
Time point:
7 d
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: Immediately
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: 1 hour
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24 h
Score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #2
Time point:
48 h
Score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #2
Time point:
72 h
Score:
1
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #2
Time point:
7 d
Score:
0
Irritation parameter:
overall irritation score
Basis:
animal: 1 and 2
Time point:
other: 24 and 72 Hours
Score:
15
Reversibility:
fully reversible within: 7 days
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 24 and 72 hours
Score:
3.8
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
The test item produced a primary irritation index of 3.8 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.
Well-defined erythema and slight edema were noted at both treated skin sites at the 24 and 48 Hour observations with well-defined erythema and very slight to slight edema noted at the 72 Hour observation.
Both treated skin sites appeared normal at the 7-Day observation.
Other effects:
- Other adverse local effects: Loss of skin flexibility and/or elasticity was noted at both treated skin sites at the 48 and 72 Hour observations. Both treated skin sites appeared normal at the 7-Day observation.
- Other adverse systemic effects: One animal showed body weight loss and the other animal showed expected gain in body weight during the study.

Orange colored staining of the test site, not preventing evaluation of skin responses, was noted at both treated skin sites during the study. The test substance is a dark orange powder.

Individual Skin Reactions

Skin Reaction

Observation Time
(following patch removal)

Individual Scores

Total

Rabbit Number and Sex

75409Male

75412Male

Erythema/Eschar Formation

Immediately

0STA

0STA

(0 )

1 Hour

0STA

0STA

( 0 )

24 Hours

2STA

2STA

4

48 Hours

2STALe

2STALe

( 4 )

72 Hours

2STALeLf

2STALeLf

4

7 Days

0

0

( 0 )

Edema Formation

Immediately

0

0

( 0 )

1 Hour

0

0

( 0 )

24 Hours

2

2

4

48 Hours

2

2

( 4 )

72 Hours

2

1

3

7 Days

0

0

( 0 )

Sum of 24 and 72‑Hour Readings (S)            :              15

Primary Irritation Index (S/4)                          :              15/4 = 3.8

Classification                                                      :              MODERATEIRRITANT


(   ) =    Total values not used for calculation of primary irritation index

STA =   Orange colored staining of the test site

Le =      Loss of skin of elasticity

Lf =       Loss of skin flexibility

Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 7

75409Male

2.73

2.84

0.11

75412Male

2.53

2.50

-0.03

Interpretation of results:
GHS criteria not met
Conclusions:
The test item produced a primary irritation index of 3.8 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted. however, the test item was classified as non irritant according to the Globally Harmonized System of Classification and Labelling of Chemicals, since the effects found were fully reversible by the end of 7 days and mean scores for erythema and edema were <2.3.

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 two rabbits produced well-defined erythema, slight edema and loss of skin elasticity and flexibility. Both treated skin sites appeared normal at the 7‑Day observation.

Conclusion

The test item produced a primary irritation index of¨3.8 and was classified as amoderateirritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted. However, the test item was classified as non irritant according to the Globally Harmonized System of Classification and Labelling of Chemicals, since the effects found were fully reversible by the end of 7 days and mean scores for erythema and edema were <2.3.

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:
The study was conducted on 22 March 2016
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Lot.Batch number: BOP 02-15 (BS-GJC 1130)
- Expiration date of the lot/batch: 18 March 2020
- Purity: 82.7% all organic components
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Local abattoir
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): typically 12 to 60 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
physiological saline
Controls:
yes
yes, concurrent vehicle
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20% w/v solution in 0.9% sodium chloride
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
3
Details on study design:
Study Design
Preparation of Corneas All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A posttreatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
Irritation parameter:
in vitro irritation score
Value:
5.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item exhibited light orange staining post treatment. The corneas treated with the negative control item were clear post treatment.
The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score The In Vitro irritancy scores are summarized as follows:

 Treatment  In vitro Irritancy Score
 Test Item  5.5
 Negative Control  1.0
 Positive Control  72.7
Interpretation of results:
GHS criteria not met
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS  Classification
 ≤ 3  No category. Not requiring classification to UN GHS or EU CLP
 > 3; ≤55  No prediction of eye irritation can be made
 > 55  Category 1. UN GHS or EU CLP Causes serious eye damage

Results

The In vitro irritancy scores are summarized as follows:

 Treatment  In vitroIrritancy Score
 Test Item  5.5
 Negative Control  1.0
 Positive Control  72.7

Conclusion

No prediction of eye irritaion can be made.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the results of skin and eye irritation studies, FAT 40871 does not warrant classification according to CLP (regulation 1272/2008) criteria.