Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin Irritation
Target Substance - Slight irritation on the 2 animal test but insufficient for classification. Human test negative.
GHS EU: NA
GHS UN: NA

Eye Irritation

OECD 437 BCOP

Target Substance - Non-Irritating.

GHS EU: NA
GHS UN: NA

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:
weight of evidence
Study period:
13 August 1979 to 26 September 1979
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Older study conducted prior to development of GLP and current test guideline; methodology is not comparable to current guidelines.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was conducted prior to adoption of OECD test guidelines. A single semi-occluded 4-hour topical application was made to the dorsum of eight rabbits; the reactions were assessed at 24, 48 and 72 hours post application. Whilst the application method is similar to current guidelines, a non-standard scoring system was used and observations were not made beyond 72 hours, therefore this study is not considered to be comparable to current guidelines.
GLP compliance:
no
Remarks:
Conducted prior to development of GLP
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9-12 weeks
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
0.5 mL of undiluted test substance was applied to the clipped dorsum of each rabbit.
Duration of treatment / exposure:
4 hours
Observation period:
Observations were made immediately after removal of the patch, and 24, 48 and 72 hours after application.
Number of animals:
Eight rabbits (one test substance and three reference controls applied to randomised sites on the rabbits).
Details on study design:
TEST SITE:
- Area of exposure: 2.5 x 2.5 cm patch on dorsum
- Type of wrap if used: The patches were prepared by attaching a piece of thin flexible polythene (3 cm x 3 cm) to a piece of zinc oxide plaster (9 cm x 2.5 cm). A 2.5 cm square of cotton gauze (8-ply folded in two) was placed onto the polythene so that the edges of the pad were attached to the plaster. The test substance was applied directly to the dry patch. The patches were firmly attached and the animals immobilised during exposure.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treatment sites were wiped clean of excess test substance at patch removal
- Time after start of exposure: 4 hours

SCORING SYSTEM: An eight point anchored ordinate scale was used (see below). The test sites were scored for erythema, oedema, cracking, scaling, scabbing, presence of exudate and haemorrhage.
a = marginal/very slight; 1 point
b = slight; 2 points
c = fairly distinct; 3 points
d = quite distinct; 4 points
e = becoming well developed; 6 points
f = well developed; 8 points
g = becoming severe; 10 points
h = severe; 12 points
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
31
Max. score:
288
Reversibility:
not specified
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
25
Max. score:
288
Reversibility:
not specified
Irritant / corrosive response data:
Marginal/slight erythema was observed in 3 animals at patch removal; 3 animals exhibited slight erythema whilst 2 animals exhibited fairly distinct erythema. Four animals exhibited marginal/very slight oedema at this time point, with 3 animals exhibiting slight oedema and the 1 remaining animal exhibiting fairly distinct oedema.
The test site of 1 rabbit had returned to normal 24 hours after application. Six rabbits exhibited marginal/very slight to slight erythema and oedema at 24 hours, the remaining rabbit exhibited erythema that was 'becoming well developed' and quite distinct erythema.
By the 48 hour observation, a second rabbit was no longer displaying signs of irritation. Five rabbits were displaying marginal/very slight erythema, and no reaction to marginal/very slight oedema. One of these rabbits also displayed marginal/very slight cracking of the skin. The remaining rabbit exhibited quite distinct erythema and 'becoming well developed' oedema.
At the 72 hour observation, 5 rabbits were free of erythema and oedema however 3 of these rabbits exhibited marginal/very slight cracking of the skin. Two rabbits still exhibited marginal/very slight erythema and oedema, with one rabbit exhibiting quite distinct erythema and oedema.
Other effects:
Pale yellow discolouration was observed in 5 animals at patch removal. The irritant response produced by the test substance, aurantiol pure, was greater than that produced by diethyl phthalate, but less than that produced by cyclamen aldehyde and geraniol.

Table 1: Skin irritation results

Rabbit No.

Time after application

4 hours (patch removal)

24 hours

48 hours

72 hours

E

O

C

E

O

C

E

O

C

E

O

C

1

b*

a

-

a

-

-

-

-

-

-

-

a

2

b*

b

-

-

-

-

-

-

-

-

-

-

3

c*

c

-

b

b

-

a

-

-

-

-

-

4

a*

b

-

a

a

-

a

a

-

-

-

a

5

c*

b

-

e

d

-

d

e

-

d

d

a

6

a

a

-

b

b

-

a

a

-

a

-

-

7

a

a

-

b

a

-

a

-

a

-

-

a

8

b

a

-

b

a

-

a

a

-

a

a

-

E = erythema; O = oedema; C = cracking.

* = pale yellow discolouration of test site; - = no reaction observed

Scoring System:

a = marginal/very slight = 1

b = slight = 2

c = fairly distinct = 3

d = quite distinct = 4

e = becoming well developed = 6

f = well developed = 8

g = becoming severe = 10

h = severe = 12

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Aurantoin caused slight irritation in rabbits following a 4-hour semi-occlusive exposure but insufficient for classification.
Executive summary:

The skin irritation potential of aurantiol pure was evaluated in New Zealand White rabbits. The study design and scoring method are not comparable to current OECD test guidelines. The test substance was applied to the clipped dorsal skin of 8 rabbits, and held in place under a semi-occlusive dressing for four hours. Each rabbit also received applications of 3 reference substances, simultaneously (diethyl phthalate, cyclamen aldehyde and geraniol). After the exposure period, the patches were removed and residual test substance was removed by wiping. Skin reactions were scored immediately after patch removal, then at 24, 48 and 72 hours after application. Reactions (erythema, oedema, cracking, scaling, scabbing, exudant and haemorrhage) were graded according to an 8-point anchored ordinate scale, ranging from ‘very slight’ to ‘severe’. The test substance caused marginal/slight to slight erythema in 7 rabbits, with 1 rabbit exhibiting quite distinct to becoming well developed erythema and oedema. Cracking of the skin of the test site was observed in 1 rabbit at the 48 hour observation, and in 4 rabbits at the 72 hour observation. With the exception of 1 rabbit, reactions appeared to ameliorate between the 24 and 72 hour observations. Erythema and oedema persisted in 3 rabbits at the 72 hour time point; as no further observations took place it is not possible to conclude on reversibility of reactions. The irritant response produced by the test substance, aurantiol pure, was greater than that produced by diethyl phthalate, but less than that produced by cyclamen aldehyde and geraniol. It can be concluded that the test substance was slightly irritating to rabbit skin.

Endpoint:
skin irritation: 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:
weight of evidence
Study period:
The study was conducted between 7 December and 17 December 1981
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Older study conducted prior to development of GLP and current test guideline; methodology is not comparable to current guidelines.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was conducted prior to development of current OECD guidelines. A preliminary irritation screen was conducted in rats, prior to a phototoxicity study. Rats were exposed dermally to the test substance for 20 minutes, and reactions were scored at intervals up to 72 hours. The study is not considered to be comparable to current guidelines.
GLP compliance:
no
Remarks:
Conducted prior to development of GLP
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house colony
- Age at study initiation: 29 days maximum
- Weight at study initiation: 61 to 73 g
- Housing: Individually in cages with wire mesh floors
- Diet (e.g. ad libitum): Ad libitum pelleted commercial rat diet
- Water (e.g. ad libitum): Ad libitum


IN-LIFE DATES: From: To: 7th to 17th December 1981 (full test including phototoxicity component)
Type of coverage:
not specified
Preparation of test site:
clipped
Vehicle:
other: ethanol
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 100%, 30% and 10% w/v in ethanol (controls received 100% ethanol)

Dosing solutions were prepared at room temperature.
Duration of treatment / exposure:
20 minutes
Observation period:
3, 6, 24, 48 and 72 hours after application
Number of animals:
3 males and 2 females per group
Details on study design:
0.1 mL test susbtance was applied to the clipped back skin of the rats for 20 minutes. After the 20 minute exposure period the skin was wiped with ethanol. No further information on the application method is given.

SCORING SYSTEM:
0 = absent
1 = very slight
2 = slight
3 = slight to distinct
4 = distinct
5 = distinct to well developed
6 = well developed
7 = well developed to severe
8 = severe
9 = missing value
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
1.06
Max. score:
9
Reversibility:
fully reversible
Remarks on result:
other: 100%
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
1.33
Max. score:
9
Reversibility:
fully reversible
Remarks on result:
other: 100%
Irritant / corrosive response data:
Very slight to slight erythema and oedema were observed up to and including the 72 hour time point in the rats exposed to 100% and 30% test substance. Very slight erythema and oedema were observed in rats exposed to 10% test substance and 100% ethanol (vehicle control group).
Very slight to slight cracking of the skin was observed in 3 rats exposed to 100% test substance at 48 hours, and in 5 rats at 72 hours. One rat displayed exhibited very slight scaling at 48 hours; by 72 hours 3 rats exhibited slight scaling and 1 rat exhibited very slight scaling. Very slight cracking of skin and scaling was observed in 3 and 1 rats, respectively, 48 hours after exposure to 30% test substance. At the 72 hour time point 4 rats exhibited very slight cracking and two rats exhibited very slight scaling. In the group exposed to 10% test substance, 2 rats exhibited very slight cracking of the skin at 48 hours and 72 hours. These rats also exhibited very slight scaling at the 72 hour observation.
Exposure to the vehicle (ethanol) also resulted in very slight cracking at 48 hours (1 rat) and 72 hours (2 rats), and very slight scaling at 72 hours (1 rat).
Other effects:
Yellow staining was observed at the test sites.
All rats gained weight during the study period.

Mean irritation scores

Test Group

(n = 5)

Time after application

3 hours

6 hours

24 hours

48 hours

72 hours

E

O

E

O

E

O

E

O

E

O

100% test substance

0.6

1.0

1.0

0.8

0.8

0.8

1.0

1.4

1.4

1.8

30% test substance

0.4

0.8

0.4

1.0

0.4

1.0

0.6

1.2

1.0

1.4

10% test substance

0.6

1.0

0.2

1.0

0.4

0.8

0.2

1.0

0.4

1.0

Control (100% ethanol)

0.8

0.4

0.6

0.6

0.4

0.6

0.2

0.8

0.4

0.8

E = erythema, O = oedema

SCORING SYSTEM:

0 = absent

1 = very slight

2 = slight

3 = slight to distinct

4 = distinct

5 = distinct to well developed

6 = well developed

7 = well developed to severe

8 = severe

9 = missing value

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Aurantoin caused slight irritation in rats following a 20 minute dermal exposure but insufficient for classification..
Executive summary:

The skin irritation potential of aurantiol pure was evaluated in a preliminary irritation screen using groups of 5 Wistar rats. The study design and scoring method are not comparable to current OECD test guidelines. The test substance (0.1 mL) was applied to the clipped dorsal skin of each rat for 20 minutes at concentrations of 100% (undiluted), 30% and 10% (in ethanol). A fourth group were exposed to the vehicle alone (100% ethanol). After the 20 minute exposure, the application sites were wiped with ethanol. Reactions were scored at 3, 6, 24, 48 and 72 hours after application, according to a 9-point scoring system. Exposure to the 100% and 30% test substance concentrations resulted in very slight to slight erythema and oedema, which persisted for up to 72 hours after application. Exposure to the 10% test substance concentration and vehicle-control (100% ethanol) resulted in very slight erythema and oedema which persisted for up to 72 hours after application. Cracking and scaling of the test sites was also observed in some animals of all treated groups. It can be concluded that 100% aurantiol pure is slightly irritating to rat skin following a 20 minute exposure. Irritant reactions to the 30% and 10% concentrations appeared reduced compared to the undiluted substance, however it should be noted that the vehicle used also caused slight irritation.

Endpoint:
skin irritation: in vivo
Remarks:
other: human volunteer
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1971
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Very brief report of a preliminary study conducted as part of another study. The study was conducted prior to development of GLP and current test guidelines.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
A skin sensitisation study was conducted in human volunteers. Skin irritation data are available from pre-testing conducted prior to the main maximisation test. Details on methodology are minimal.
GLP compliance:
no
Remarks:
Conducted prior to development of GLP
Specific details on test material used for the study:
Tested as is.
Species:
other: Human subjects
Strain:
other: Patients
Details on test animals or test system and environmental conditions:
Healthy human adult male subjects
Type of coverage:
occlusive
Preparation of test site:
other: none required
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
No information available
Duration of treatment / exposure:
48 hours
Observation period:
No information available
Number of animals:
Ten
Details on study design:
A patch containing the test substance was applied to normal skin on the back of each subject for 48 hours under occlusion.
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
8
Reversibility:
not specified
Remarks on result:
other: 3 page document only.
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
8
Reversibility:
not specified
Remarks on result:
other: 3 page document only.

There were no irritant reactions following a 48 hour occluded exposure to the test substance.

Interpretation of results:
GHS criteria not met
Conclusions:
There were no irritant reactions following a 48 hour occluded exposure to the test substance on human subjects.
Executive summary:

A preliminary irritation screen was conducted in 10 healthy adult human male volunteers, as part of a skin sensitisation maximisation test. The test substance was applied undiluted to normal skin on the back of the subjects, and held in place for 48 hours under an occlusive dressing. No irritant reactions were observed following exposure (the time at which observations were made was not stated).

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:
February 2014
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 Ocular Corrosives and Severe Irritants)
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)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Description: Yellow viscous liquid
Batch: SC00010629
Purity/Composition: UVCB substance
Species:
other: Bovine eyes
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained direct from the slaughterhouse. Eyes were from young cattle slaughtered at Vitelco's Hertogenbosch, The Netherlands. The eyes were excised as soon as possible after slaughter, and used on the same day. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. Prior to use, the corneas were checked for unacceptable defects such as opacity, scratches, pigmentation and neovascularization by submersing them in physiological saline and holding them in the light. Those exhibiting defects were discarded.

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL per cornea (no corrections were made for the purity/composition of the test substance)

Duration of treatment / exposure:
10 ± 1 minutes
Observation period (in vivo):
Opacity was determined following a 120 ± 10 minute incubation period.
Number of animals or in vitro replicates:
Three corneas were used for each group.
Details on study design:
PREPARATION AND SELECTION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 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. 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. 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
The medium from the anterior compartment was removed and 750 µl of either the negative control (physiological saline) or positive control (10% (w/v) Benzalkonium Chloride) was introduced onto the epithelium of the cornea. The test substance was introduced onto the epithelium of the cornea with a cut off plastic pipette, enough to cover the epithelium completely. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

OPACITY MEASUREMENT
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in 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 substance 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 substance 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.

SODIUM FLUORESCEIN APPLICATION
Following the final opacity measurement, permeability of the cornea to Na-fluorescein 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 4 mg Na-fluorescein/mL cMEM solution. 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. 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.
Irritation parameter:
other: IVIS (in vitro irritancy score)
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
IVIS <3

The individual in vitro irritancy scores for the negative controls ranged from -0.2 to 0.3. The individual positive control in vitro irritancy scores ranged from 130 to 138 for Benzalkonium Chloride. The corneas treated with the positive control substance were turbid after the 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range, except for one cornea. The negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 133 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Table 1. Summary of opacity, permeability and in vitro scores

 

Treatment

Mean Opacity1

Mean Permeability1

Mean In Vitro Irritation Score1,2

Negative Control

0

0.000

0.0

Positive Control (Benzalkonium Chloride)

75

3.882

133

Aurantiol Pure

2

-0.032

1.5

1 Calculated using the negative control mean opacity and mean permeability values

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Interpretation of results:
GHS criteria not met
Conclusions:
The mean in vitro irritation score was 1.5, therefore the test substance is considered to be non-irritating and no classification is required.
Executive summary:

Screening for the eye irritant potential of Aurantiol Pure was conducted using the Bovine Corneal Opacity and Permeability (BCOP) test (OECD Guideline 437). The test substance was applied directly to the corneas, undiluted, and incubated for 10 minutes. Negative controls (physiological saline) and positive controls (10% w/v benzalkonium chloride) were included. Opacity determinations were performed on each cornea using an opacitometer, before and after exposure. The change in opacity for each cornea was calculated. After incubation, the medium in each treatment compartment was transferred to 96-well plates and the optical density determined at 490 nm (OD490). The in vitro irritancy score (IVIS) for each group were calculated by adding the mean opacity value to (15 x mean OD490 value). The negative control (physiological saline) 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, except the response of one cornea. This resulted in a mean permeability value just above the upper limit of the range. However since the other two values were less than the upper limit of the laboratory historical range, this deviation in the permeability score had no effect on the results of the study. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 133 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Aurantiol Pure did not cause ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment. It is concluded that, under the experimental conditions, Aurantiol Pure is a non-irritant in the BCOP test. Since the 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

Justification for classification or non-classification