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Administrative data

Description of key information

Skin irritation: Not irritating, Method equivalent/similar to OECD 404, O'Connor (1979).
Eye irritation: Not irritating, OECD 405 and EU Method B.5, Sanders (2013).

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:
13 August 1979 to 01 October 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A non-GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
A 0.5 mL aliquot of the test material was applied to the clipped dorsum of rabbits for 4 hours under a semi-occlusive patch. The irritation reactions were assessed immediately after removal of the patch and after 24, 48 and 72 hours.
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals 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:
TEST MATERIAL
- Amount applied: 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
8
Details on study design:
TEST SITE
- Area of exposure: Rabbits were exposed on their dorsum, the specific treatment site were randomised. The whole dorsal surface was clipped 3-4 days before exposure. Only animals in the telogen phase of hair growth were selected.
- Type of wrap if used: Occlusive 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 laid on the polythene such that the edges of the pad were attached to the zinc oxide plaster. The test material was applied to the dry patch. The patches were firmly attached to ensure good contact between the skin and the test material. During exposure, the animals were immobilised in a canvas body sleeve.

REMOVAL OF TEST SUBSTANCE
- Washing: The test site was wiped clean of excess test material.
- Time after start of exposure: 4 hours, immediately after patch removal.

REFERENCE STANDARDS
- Diethyl phthalate
- Cyclamen aldehyde
- Geraniol
These standards are perfumery materials that are regularly used as marker controls in skin irritation tests.
All three standards were applied to each of the test rabbits, alongside the test material. Application sites were randomised on the different animals.

SCORING SYSTEM: The skin sites were assessed for irritation immediately after treatment, and at 24, 48 and 72 hours. Each animal was scored blind and the scores for each skin site were not linked to the material tested until the scoring at 72 hours was completed. The sites were scored for erythema, edema, cracking and scaling and any other feature using an 8-point anchored ordinate scale ranging from "a" (very slight) to "h" (severe) see Table 1. At the end of the test the reaction grades for each treatment group were converted to numerical scores which were used to calculate the total irritation score per site.
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24 hours
Score:
0.5
Max. score:
12
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24 hours
Score:
0.25
Max. score:
12
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 48 hours
Score:
0.375
Max. score:
12
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 48 hours
Score:
0.375
Max. score:
12
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 72 hours
Score:
0.375
Max. score:
12
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 72 hours
Score:
0.25
Max. score:
12
Reversibility:
not fully reversible within: 72 hours
Irritant / corrosive response data:
A single application of test material produced marginal erythema in some animals and marginal edema in a few animals at 24 hours. By 48 hours and 72 hours there was marginal erythema and edema in a few animals. The response was broadly similar to that produced by the control, diethyl phthalate, but considerably less than that produced by the controls, cyclamen aldehyde and geraniol. Total irritation scores of the test material and standards for comparison are given in Table 2, where Table 3 shows the macroscopic findings.

Table 2: Total Irritation Scores

Rabbit No. Irritation scores
Diethyl Phthalate Test Material Cyclamen Aldehyde Geraniol
1216 0 0 23 32
1217 3 5 31 34
1218 2 3 30 28
1219 4 2 26 26
1220 4 3 35 22
1221 2 0 31 34
1224 2 6 27 38
1244 2 9 41 30
         
Overall irritation score 19 28 244 244
Mean score per site 2.38 4.0 30.5 30.5
Mean score per site per day 0.59 1.0 7.63 7.63
Mean irritation ranking 1.44 1.56 3.44 3.56

Table 3. Macroscopic Findings For Sites Exposed to the Test Material

Rabbit No. 4 Hours 24 Hours 48 Hours 72 Hours Totals Total Score
Erythema Edema Erythema Edema Erythema Edema Erythema Edema Erythema Edema
1216 0 0 0 0 0 0 0 0 0 0 0
1217 1 1 1 0 1 0 1 0 4 1 5
1218 1 1 1 0 0 0 0 0 2 1 3
1219 1 1 0 0 0 0 0 0 1 1 2
1220 1 2 0 0 0 0 0 0 1 2 3
1221 0 0 0 0 0 0 0 0 0 0 0
1224 0 0 1 1 1 1 1 1 3 3 6
2244 1 1 1 1 1 2 1 1 4 5 9
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the test material elicited only very minor reactions in a few of the animals during the course of the study. The test material was concluded to be very slightly irritating. The persistency of this slight irritation continued until termination at 72 hours, no further observations were made.
Executive summary:

The skin irritation potential of the test material was investigated according to a methodology similar to that of OECD 404. During the study 0.5 mL of the undiluted test material was applied to the clipped dorsum of 8 rabbits for 4 hours under a semi-occlusive patch. The irritation reactions were assessed immediately after removal of the patch and after 24, 48 and 72 hours.

During the course of the study a single application of test material was seen to produce ‘marginal’ erythema and ‘slight’ edema in five out of eight animals at 24 hours. By 48 hours and 72 hours ‘marginal’ erythema and edema was observed in between two and four out of eight animals. The scores assigned in this study are roughly equivalent to a score of 1 according to the Draize scale (1977).

The response was broadly similar to that produced by the control, diethyl phthalate, but considerably less than that produced by the positive controls, cyclamen aldehyde and geraniol.

Under the conditions of the study, the test material was considered to be slightly irritating; however the elicited reaction was below the limits of classification.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2012 to 04 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: Twelve to twenty weeks old
- Weight at study initiation: 2.98 to 3.42 kg
- Housing: Individually housed in suspended cages
- Diet: 2930C Teklad Global Certified Rabbit diet ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Humidity: 30-70%
- Air changes: At least fifteen changes per hour
- Photoperiod: 12 hours light/12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Administration of the test material: A single treatment was administered to initially one rabbit. 0.1 mL of the test material was instilled into the conjunctival sac of the right eye. The eyelids were held together for a second to prevent loss of the test material. The left eye remained untreated as a control. Based on the initial pain reaction, the second rabbit was treated using the same procedure as the first animal after the administration of a local anaesthetic instilled into both eyes of the second rabbit.
Duration of treatment / exposure:
A single treatment was administered, no washing was performed
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
2
Details on study design:
SCORING SYSTEM:
Draize Scale for Scoring Ocular Irritation

TOOL USED TO ASSESS SCORE: Standard ophthalmoscope

OTHER:
- pH: Prior to the commencement of the study, the pH of the test material was determined undiluted and as a 90 % aqueous preparation. The pH of the test material was determined to be approximately pH 7 undiluted, and approximately pH 6 as a 90 % aqueous preparation.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Basis:
animal: 72444
Time point:
other: 24 hours
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal: 72515
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritant / corrosive response data:
No corneal or iridial effects were noted in either animal at any of the time points during the study. Both animals had minimal conjunctival irritation at one hour post treatment. This had completely cleared in one animal by the 24 hour observation time, minimal irritation was still noted in the other animal at the 24 hour observation time, but had resolved by 48 hours.
Other effects:
Both animals employed in the study demonstrated expected bodyweight gain during the course of the study.

Table 2: Individual irritation scores

Rabbit Number and Sex

72444 Male

72515 Male

Initial Pain Reaction

3

0+

Time After Treatment

(hours)

1

24

48

72

1

24

48

72

Cornea

0

0

0

0

0

0

0

0

Degree of opacity

0

0

0

0

0

0

0

0

Area of Cornea Involved

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

Conjunctivae

0

0

0

0

0

0

0

0

Redness

1

1

0

0

1

0

0

0

Chemosis

1

0

0

0

1

0

0

0

Discharge

0

0

0

0

0

0

0

0

+ Local anaesthetic (one drop of 0.5 % Tetracaine hydrochloride)

 

Table 3: Bodyweights and bodyweight changes

Rabbit Number and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 3

72444 Male

3.42

3.5

0.08

72515 Male

2.98

3.03

0.05

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the test material induce only minimal reversible irritation during the test in the New Zealand White rabbit. The test material was therefore considered to be not irritating.
Executive summary:

The irritancy potential of the test material to the eye of the New Zealand White rabbit was evaluated in a GLP compliant study designed to be compatible with OECD 405 and EU Method B.5. Undiluted test material (0.1 mL) was instilled into the eye of two rabbits. After the initial pain reaction of the first rabbit, the test in the second rabbit was performed with a local anaesthetic. The rabbits were observed at 1, 24, 48 and 72 hours for signs of ocular irritation. Minimal conjunctival irritation was noted in both animals an hour post-instillation. One treated eye appeared normal at the 24 hour observation, and the other appeared normal at the 48 hour observation. Under the conditions of the test, the test material is considered to be non-irritating to the New Zealand White rabbit eye.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

In the key study (O’Connor, 1979), the skin irritation potential of the test material was investigated according to a methodology similar to that of OECD 404. During the study 0.5 mL of the undiluted test material was applied to the clipped dorsum of 8 rabbits for 4 hours under a semi-occlusive patch. The irritation reactions were assessed immediately after removal of the patch and after 24, 48 and 72 hours. During the course of the study a single application of test material was seen to produce ‘marginal’ erythema and ‘slight’ edema in five out of eight animals at 24 hours. By 48 hours and 72 hours ‘marginal’ erythema and edema was observed in between two and four out of eight animals. The scores assigned in this study are roughly equivalent to a score of 1 according to the Draize scale (1977). The response was broadly similar to that produced by the control, diethyl phthalate, but considerably less than that produced by the positive controls, cyclamen aldehyde and geraniol. Under the conditions of the study, the test material was considered to be slightly irritating; however the elicited reaction was below the limits of classification. The study was assigned a reliability score of 1 in accordance with principles defined in Klimisch (1997).

Two additional studies have been provided as supporting evidence Mulhern et al. (1990) and Moreno (1973). Mulhern et al. (1990) assessed dermal irritation as a result of exposure to the test material as part of a repeat dose dermal study, conducted following a methodology similar to the OECD guideline 411. Groups of twelve male and twelve females rats were exposed to the test material for 6 hours per day at concentrations of 100, 300 and 1000 mg/kg/day over 13 consecutive weeks. Under the conditions of the study, transient incidences of slight desquamation and erythema occurred sporadically throughout the dosing period, and were observed at a similar incidence and level of severity in all groups including the control. Since no dose response correlation was observed, and since similar skin assessments were made on the animals in the control group, the test material was considered to be not irritating.

The second supporting study Moreno, (1973) discussed dermal irritation as part of an acute dermal toxicity study in rabbits. The dermal irritation data was reported with limited details. Ten rabbits were subjected to dermal exposure at 5000 mg/kg test material. Following treatment, the animals were observed for mortality, clinical signs of toxicity and dermal irritation. The report stated that 3 out of 10 animals exhibited slight redness and 1 out of 10 animals exhibited moderate redness, in response to the acute exposure. Based on the available information the test material was considered to be non-irritating.

Eye Irritation

The key study, Sanders (2013) investigated the irritancy potential of the test material to the eye of the New Zealand White rabbit was evaluated in a GLP compliant study designed to be compatible with OECD 405 and EU Method B.5. Undiluted test material (0.1 mL) was instilled into the eye of two rabbits. After the initial pain reaction of the first rabbit, the test in the second rabbit was performed with a local anaesthetic. The rabbits were observed at 1, 24, 48 and 72 hours for signs of ocular irritation. Minimal conjunctival irritation was noted in both animals an hour post-instillation. One treated eye appeared normal at the 24 hour observation, and the other appeared normal at the 48 hour observation. Under the conditions of the test, the test material is considered to be non-irritating to the New Zealand White rabbit eye.

Two studies have been provided as supporting data, Warren (2012) and Hopf (1968). Warren (2012), a complete study testing eye irritation in vitro, and Hopf (1968) a short description of an in vivo study which reports both support the conclusions of the key study, Sanders (2013).

Warren (2012) outlines the investigation into the eye irritation potential of the test material in vitro using the SkinEthic reconstructed Human Corneal Epithelium model after a treatment period of 10 minutes. The SkinEthic model consists of transformed human corneal epithelial cells of the cell line HCE that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. During the study, the test material was applied directly to the culture surface, at the air interface, so that undiluted test material could be tested directly. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test material treated tissues relative to the negative control. One tissue for each treatment group was retained for possible tissue histopathology. Under the conditions of the study, the test material did not directly reduce MTT and the relative mean viability of the test material treated tissues after a 10 minute exposure period was calculated to be 108.0 % indicating that the test material is a non-irritant. It was considered unnecessary to proceed with tissue histopathology. The study was performed to a good standard with complete reporting; however the study followed a non-validated guideline. Accordingly the study was assigned a reliability score of 2 according to the criteria for assessing data quality defined in Klimisch (1997).

Hopf (1968) reports the potential of the test material to cause irritation to the eye assessed according to the Draize method. During the study the conjunctival sac of eight rabbits were administered with 0.1 mL of test material diluted 1:1 with olive oil. After one minute fixation, the eyes of four of the animals were rinsed with warm water; the eyes of the remaining animals were left with the dose in situ. Following administration the rabbits were observed for any signs of mucosal reactions or immune response, as determined by the scoring system of Draize. Under the conditions of the study, no mucosal reactions were observed in the area of the eye or mucous membranes of the eyes. In addition, no spontaneous immune responses were noted. The test material is therefore considered to be non-irritating to the eyes. The study was presented in very limited detail. It was not possible to assess the quality of the data and was therefore assigned a reliability score of 4 according to Klimisch (1997).


Justification for selection of skin irritation / corrosion endpoint:
The non-GLP key study was performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. Accordingly it was assigned a reliability score of 2 in line with the criteria defined by Klimisch (1997) and considered suitable as an accurate reflection of the test material.

Justification for selection of eye irritation endpoint:
The key study was performed in line with GLP and currently accepted in vivo standard testing guidelines. The study was complete and performed to a high standard and the results were reported to a high level of detail. The study was therefore assigned a reliability score of 1 in accordance with the principles for assessing data quality as described in Klimisch (1997). The study was therefore considered to be an accurate reflection of the effects of the test material, and the results of the study were considered to be suitable to be taken forward for risk assessment and classification and labelling purposes.

Justification for classification or non-classification

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, the substance does not meet the criteria for classification as a skin or eye irritant.