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Administrative data

Description of key information

Skin irritation

In a K2 in vivo skin irritation study in New Zealand White rabbits according to OECD Guideline 404 and EU method B.4, no evidence for skin irritation was noted for T001325.

Eye irritation

In a K1 Bovine Corneal Opacity and Permeability test, performed according to OECD guideline 437 and EU Method B.47, T001325 did not induce ocular irritation. No classification was required for eye irritation or serious eye damage.

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:
2003-07-07 to 2004-09-22
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study is similar to OECD Guideline 404 (Acute Dermal Irritation / Corrosion) and EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion);
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
no
Remarks:
Study was conducted in a facility operating to Good Laboratory Practice within the UK national GLP monitoring programme. No formal claim of GLP compliance was made for this study.
Specific details on test material used for the study:
no data
Species:
rabbit
Strain:
New Zealand White
Remarks:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: From: 2003-07-07 To: 2003-07-10
Type of coverage:
semiocclusive
Preparation of test site:
other: intact
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): not applicable
Duration of treatment / exposure:
A single 4 hour treatment
Observation period:
1, 24, 48, and 72 hours
Number of animals:
three male rabbits
Details on study design:
TEST SITE
- Area of exposure: no data
- % coverage: no data
- Type of wrap if used: no data


REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data
- Time after start of exposure: no data


SCORING SYSTEM:
Animals were scored on erythema/eschar and oedema formation.
Primary Irritation Index was assigned as follows:
0 = Non-irritant
> 0 - 2 = Mild irritant
>2 - 5 = Moderate irritant
>5 - 8 = Severe irritant
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 24 and 72 hours
Score:
0
Max. score:
8
Reversibility:
other: Non-irritating so not applicable
Remarks on result:
other: Non-irritant
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 93
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: male
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 94
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: male
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal 96
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: male
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 93
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: male
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 94
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: male
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal 96
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: male
Irritant / corrosive response data:
Non-irritant
Other effects:
Light brown-coloured staining was reported for all (3) test animals.

Skin Reaction

Observation Time

Rabbit 1

Rabbit 2

Rabbit 3

Total

Erythema/ Eschar Formation

1 Hour

0 STA

0 STA

0 STA

( 0 )

24 Hours

0 STA

0 STA

0 STA

0

48 Hours

0 STA

0 STA

0 STA

( 0 )

72 Hours

0 STA

0 STA

0 STA

0

Total

0

0

0

0

Mean Score

0.0

0.0

0.0

0.0

Oedema Formation

1 Hour

0

0

0

( 0 )

24 Hours

0

0

0

0

48 Hours

0

0

0

( 0 )

72 Hours

0

0

0

0

Total

0

0

0

0

Mean Score

0.0

0.0

0.0

0.0

Sum of 24 and 72 Hour Readings (s)

0

Primary Irritation Index (S/6) and Classification

0/6 = 0.0
NON-IRRITANT

 

STA = Light brown-coloured staining

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

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was found to have a score of 0 for erythema/eschar formation and oedema formation at 1, 24, 48, and 72 hours. The substance was found not to be irritating. The test material did not meet the criteria for classification as irritant or corrosive according to EU labelling regulations Commission Directive 2001/59/EC.
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:
From 19 October 2015 to 27 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study according to OECD guideline 437 and EU method B.47.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Principles of method if other than guideline:
The study procedures were also in compliance with the following guidelines:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P, Dukic M, Alix D and Sina J F, Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15FB2768
- Expiration date of the lot/batch: 2017-07-01 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: a 20% (w/v) suspension of the test item was prepared in physiological saline. The formulation was treated with ultrasonic waves to obtain a homogeneous suspension.

FORM AS APPLIED IN THE TEST (if different from that of starting material): 20% (w/v) suspension in physiological saline
Species:
other: bovine eyes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible but within 4 hours after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.

- Preparation of corneas: the eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

- Cornea selection and opacity reading: after the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

- Treatment of corneas and opacity measurements: The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) test item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.
Vehicle:
physiological saline
Controls:
other: negative control (physiological saline) and positive control (20% (w/v) Imidazole solution in physiological saline)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20% (w/v) suspension, the formulation was treated with ultrasonic waves to obtain a homogeneous suspension

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/v) imidazole solution
Duration of treatment / exposure:
Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C
Observation period (in vivo):
Cornea selection and opacity reading:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
Opacity measurements:
After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group.
Details on study design:
OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (l = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the devide OP-KIT) was calculated according to:
opacity = ((I0/I)-0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea before/after test item treatment.
The change of opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

APPLICATION OF SODIUM FLUORESCEIN
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

ELECTRONIC DATA CAPTURE
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Value:
0.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range: -0.7 to 0.7
Irritation parameter:
cornea opacity score
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range -0.9 to 0.6
Irritation parameter:
other: permeability score
Value:
0.011
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Range: 0.008 to 0.016
Other effects / acceptance of results:
mean in vitro irritancy scores (range):
negative control: -1.0 (-1.1 to -0.9)
positive control: 109.8 (91.2 to 119.2)
test item: 0.2 (-0.7 to 0.7)

mean opacity scores (range):
negative control: -1.1 (scores for 3 corneas were -1.1)
positive control: 89.0 (79.8 to 96.6)
test item: 0.0 (-0.9 to 0.6)

mean permeability scores (range):
negative control: 0.004 (0.000 to 0.010)
positive control: 1.387 (0.755 to 1.915)
test item: 0.011 (0.008 to 0.016)

The corneas treated with the positive control were turbid after the 240 minutes of treatment.
No pH effect of the test item was observed on the rinsing medium.

Due to a high response of the negative control opacity and test item results spread over 2 categories, the first test was rejected and a repeat test was performed. The results reported in this endpoint study record were obtained in the repeat test.

Interpretation (repeat test):

The IVIS of all replicates was within one category.

Discussion:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 109.8 (91.2 to 119.2) and within the laboratory historical positive control data range. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoint, resulting in a mean in vitro irritancy score of 0.2 (-0.7 to 0.7) after 240 minutes of treatment.

Since the test item induced an IVIS <=3, no classification is required for eye irritation or serious eye damage.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 (-0.7 to 0.7) after 240 minutes of treatment. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

An acute dermal irritation study of T001325 was performed by Sanders (2004) on New Zealand White rabbits (3 males) after 4 hours of exposure to 0,5 g of test item. Skin reactions were recorded 1, 24, 48 and 72 hours after administration and scored according to the Draize scale. No evidence of skin irritation was noted. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP Regulation (EC) No 1272/2008.

An in vitro skin irritation study was waived based on the justification that adequate data from an in vivo skin irritation study are available.

Eye irritation

Eurlings (2016) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 750 μl of T001325 (20% w/w non-homogenous suspension in physiological saline) was applied for 240 minutes on 3 corneas. Both opacity and permeability were measured and the resulting objective values were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 (-0.7 to 0.7) after 240 minutes of treatment.

Since the test item induced an IVIS <3, it is concluded that no classification is required for T001325 for eye irritation or serious eye.

In addition, a Rabbit Enucleated Eye Test (REET) was performed by Sanders (2004) to assess the ocular irritancy potential of T001325. Three enucleated eyes of New Zealand White rabbits were treated with 0.1 mL of T001325. Corneal opacity (60, 120, 180 and 240 minutes after application), corneal swelling (60, 120 and 240 minutes after application) and fluorescein uptake (240 minutes after application) were observed and scored. No indication of irritation was noted. The test item was considered unlikely to have the potential to cause severe ocular irritancy in-vivo.

Justification for classification or non-classification

Skin irritation:

According to the in vivo acute dermal irritation study, no evidence for skin irritation was noted for T001325. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP Regulation (EC) No 1272/2008.

Eye irritation:

According to the in vitro eye irritation study (BCOP), T001325 did not induce ocular irritation. The test item did not meet the criteria for classification for eye irritation or serious eye damage according to UN GHS Classification