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Description of key information

Non skin irritating.

Eye Irritating.

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:
October from 19th to 26th, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: approximately twelve to sixteen weeks old.
- Weight at study initiation: 2.24 - 2.37 kg
- Housing: individually housed in suspended metal cages.
- Diet: free access to food (Rabbit Diet, Preston Farmers Limited, Nerv Leake, Boston, Lincolnshire, U.K. and Spillers Rabbit Diet, Daigety Agriculture Ltd., Almondsbury, Bristol) was allowed throughout the study.
- Water: free access to mains drinking water.
- Acclimation period: minimum acclimatisation period of five days.
- Health check: on the day before the test each of a group of rabbits was clipped free of fur from the dorsal/ flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 21 °C
- Relative humidity: 47 - 65 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: 12 hours light and 12 hours darkness.
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.5 g of test material were moistened with 0.5 ml of distilled water
Duration of treatment / exposure:
4 hours
Observation period:
7 days
Number of animals:
1 female and 2 males
Details on study design:
TEST SITE
- Area of exposure: test item was introduced under a 2.5 cm × 2.5 cm gauze patch and placed in position on the shorn skin.
- Type of wrap if used: the patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm × 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing: residual test material was removed by gentle swabbing with cotton wool soaked in diethyl ether.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
Approximately one hour following the removal of the patches and 24, 48 and 72 hours later, the test sites were examined. An additional observation was made on day 7 to assess the reversibility of the skin reactions.

SCORING SYSTEM
The test sites were examined for evidence of primary irritation and scored according to scale decribed in the Draize J.H. (1959), Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the safety of chemicals in Foods, Drugs and Cosmetics".

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 slight eschar fornation (injuries in depth) 4

Oedema formatíon
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4
Irritation parameter:
erythema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Reversibility:
fully reversible within: 24 hours
Irritant / corrosive response data:
Faint yellow-coloured staining caused by the test material was noted at two treated skin sites during the study. It did not affect evaluation of skin responses.
Very slight erythema was noted at all treated skin sites 1, 24 and 48 hours after patch removal and continued to be noted at two treated skin sites at the 72-hour observation.
Very slight oedema was noted at two treated skin sites one hour after patch removal.
All treated skin sites appeared normal at the 7-day observation.
The test material produced a primary irritation index of 0.8 and was classified as a mild irritant to rabbit skin. No corrosive effects were noted.

Individual reactions

Animal  Reaction 1 hrs 24 hrs 48 hrs 72 hrs 7 days Mean 24/48/72 hrs
28 female Erythema/Eschar 1 STA 1 STA 1 STA 1 STA 0 1.00
39 male Erythema/Eschar 1 1 1 0 0 0.67
68 male Erythema/Eschar 1 STA 1 STA 1 STA 1 STA 0 1.00
28 female Oedema 1 0 0 0 0 0.00
39 male Oedema 0 0 0 0 0 0.00
68 male Oedema 1 0 0 0 0 0.00

STA: faint yellow-coloured staining caused by the material

Interpretation of results:
other: not classified, according to the CLP Regulation (EC 1272/2008)
Conclusions:
Not irritating
Executive summary:

The study was performed to assess the irritancy potential of the test material following a single, 4-hour, semi-occluded application to the intact rabbit skin. The method used followed OECD guideline 404.

Faint yellow-coloured staining caused by the test material was noted at two treated skin sites during the study. It did not affect evaluation of skin responses.

Very slight erythema was noted at all treated skin sites 1, 24 and 48 hours after patch removal and continued to be noted at two treated skin sites at the 72-hour observation. Very slight oedema was noted at two treated skin sites one hour after patch removal. All treated skin sites appeared normal at the 7-day observation.

The test material produced a primary irritation index of 0.8 and was classified as a mild irritant to rabbit skin. No corrosive effects were noted.

Conclusion

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions.

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:
November from 05th to 19th, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
treated eye not rinsed
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: approximately twelve to sixteen weeks old.
- Weight at study initiation: 2.72 - 2.86 kg
- Housing: individually housed in suspended metal cages.
- Diet: free access to food (Rabbit Diet, Preston Farmers Limited, Nerv Leake, Boston, Lincolnshire, U.K.l) was allowed throughout the study.
- Water: free access to mains drinking water.
- Acclimation period: minimum acclimatisation period of five days.
- Health check: immediately before the start of the test, both eyes of the three provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Animals showing evidence of ocular Iesions were rejected and replaced.

ENVIRONMENTAL CONDITIONS
- Temperature: 16 - 24 °C
- Relative humidity: 51 - 72 %
- Air changes: approximately 15 changes per hour.
- Photoperiod: 12 hours light and 12 hours darkness.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 ml (approx. 96 mg)
Observation period (in vivo):
14 days
Number of animals or in vitro replicates:
One rabbit was initially treated; after consideration of the ocular responses produced in the first treated animal, two additional animals were treated.
1 male and 2 females
Details on study design:
TREATED EYES
In order to minimise pain on instillation of the test material, one drop of local anaesthetic "Ophthaine", 0.5 % proxymetacaine hydrochloride was instilled into both eyes of these animals 1-2 minutes before treatment.

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by use of the light source from a standard ophthalmoscope.

SCORING SYSTEM
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation described in Draize J.H. 1959, Association of Food and Drug Officials of the united States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics"). Any other adverse ocular effects were also noted.
Additional observations were made on days seven and fourteen to assess the reversibility of the ocular effects.

CORNEA:
A Opacity and degree of density (most dense area scored) .
No opacity: 0
Scattered or diffuse area, details of iris clearly visible: 1
Easily discernible translucent areas, details of iris slightly obscured: 2
Opalescent areas, no details of iris visible, size of pupil barely discernible: 3
Opaque, iris invisible: 4

B Area of cornea involved
One quarter (or less) but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4
A × B × 5; Maximum possible score = 80

IRITIS:
A Values
Normal: 0
Folds above normal, congestion, swelling, circumcornea injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive): 1
No reaction to light, hemorrhage, gross destruction (any or all of these): 2
A × 5; Maximum possible score = 10

CONJUNCTIVAE:
A Redness (refers to lids and bulbar conjunctivae excluding cornea and iris)
Vessels normal: 0
Vessels definitely injected above normal: 1
More diffuse, deeper crimson red, individual vessels not easily discernible: 2
Diffuse beefy red: 3

B Chemosis
No swelling: 0
Any swelling above normal (includes nictitating membrane): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids about half closed to completely closed: 4

C Discharge
No discharge: 0
Any amount different from normal (does not include small amounts observed in inner cantus of normal animals): 1
Discharge with moistening of the lids and hairs just adjacent to lids: 2
Discharge with moistening of the lids and hairs, and considerable area around the eye: 3
(A + B + C) × 2; Maximum possible score = 20
Irritation parameter:
cornea opacity score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
1
Reversibility:
fully reversible within: 14 days
Irritation parameter:
iris score
Basis:
animal: 2/3
Time point:
24/48/72 h
Score:
< 1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal: 2/3
Time point:
24/48/72 h
Score:
>= 2
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
animal: 2/3
Time point:
24/48/72 h
Score:
< 2
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Vocalisation was noted in one animal approximately ten seconds after dosing.
Faint yellow-coloured staining caused by the test material was noted in all treated eyes during the study. This did not prevent accurate evaluation of ocular reactions.
A dulling of the normal lustre of the corneal surface was noted in two treated eyes one hour after treatment. Areas of diffuse corneal opacity were noted in one treated eye one hour after treatment and in all treated eyes at the 24-hour observation. Diffuse corneal opacity persisted in all treated eyes up to the 72-hour observation and in one treated eye at the 7-day observation.
Iridial inflammation was noted in all treated eyes one hour after treatment, at the 24 and 48-hour observations and in one treated eye at the 72-hour observation.
Moderate to severe conjunctival irritation was noted in all treated eyes one hour after treatment with moderate conjunctival irritation at the 24 and 48-hour observations. Minimal to moderate conjunctival irritation was noted in all treated eyes at the 72-hour observation with minimal conjunctival irritation in one treated eye at the 7-day observation.
Residual test material was noted around the treated eyes of all animals during the study.
All treated eyes appeared normal 7 - 14 days after treatment.
The test material produced a maximum group mean score of 36.0 and was classified as a moderate irritant to the rabbit eye according to a modified Kay and Calandra classification system.
Animal Reaction 24 hrs 48 hrs 72 hrs 7 days 14 days Mean 24/48/72 hrs
70 male Corneal opacity 1 1 1 1 0 1.00
88 female Corneal opacity 1 1 1 0 0 1.00
92 female Corneal opacity 1 1 1 0 0 1.00
70 male Iris 1 1 1 0 0 1.00
88 female Iris 1 1 0 0 0 0.67
92 female Iris 1 1 0 0 0 0.67
70 male Conjunctival redness 2 2 2 1 0 2.00
88 female Conjunctival redness 2 2 1 0 0 1.70
92 female Conjunctival redness 3 3 1 0 0 2.33
70 male Conjunctival chemosis 3 2 2 0 0 2.33
88 female Conjunctival chemosis 2 1 0 0 0 1.00
92 female Conjunctival chemosis 2 2 1 0 0 1.67

Individual reactions

70 male 88 female 92 female
1 hr 24 hrs 48 hrs 72 hrs 7 days 14 days 1 hr 24 hrs 48 hrs 72 hrs 7 days 1 hr 24 hrs 48 hrs 72 hrs 7 days
Cornea opacity 1 1 1 1 1 0 d 1 1 1 0 d 1 1 1 0
Area 4 4 2 2 1 0 4 3 3 2 0 4 4 4 2 0
Score 20 20 10 10 5 0 0 15 15 10 0 0 20 20 10 0
Iris 1 1 1 1 0 0 1 1 1 0 0 1 1 1 0 0
Score 5 5 5 5 0 0 5 5 5 0 0 5 5 5 0 0
Conjuntival redness 2s 2 2 2 1 0 2s 2 2 1 0 2s 3 3 1 0
Conjuntival chemosis 3 3 2 2 0 0 2 2 1 0 0 3 2 2 1 0
Conjuntival discharge 3re 2re 1re 0re 0 0 3re 1re 1re 0 0 3re 2re 1re 0re 0
Score 16 14 10 10 2 0 14 10 8 2 0 16 14 12 5 0
Total score 41 39 25 25 7 0 19 30 28 12 0 21 39 37 14 0

d = dulling of the nornal lustre of the corneal surface

re = resídual test material around the eye

s = faint yellow staining

Interpretation of results:
other: Eye Irrit. 2 (H319), according to the CLP Regulation (EC 1272/2008)
Conclusions:
Eye irritant.
Executive summary:

The study was performed to assess the irritancy potential of the test material following a single application to the rabbit eye. The method used followed the recommendations of OECD guideline 405.

One rabbit was initially treated. A volume of 0.1 ml of test material, which was found to weigh approximately 96 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye. The left eye remained untreated and was used for control purposes. Immediately after administration of test substance, an assessment of the initial local pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

Vocalisation was noted in one animal approximately ten seconds after dosing. Faint yellow-coloured staining caused by the test material was noted in all treated eyes during the study. This did not prevent accurate evaluation of ocular reactions. A dulling of the normal lustre of the corneal surface was noted in two treated eyes one hour after treatment. Areas of diffuse corneal opacity were noted in one treated eye one hour after treatment and in all treated eyes at the 24-hour observation. Diffuse corneal opacity persisted in all treated eyes up to the 72-hour observation and in one treated eye at the 7-day observation. Iridial inflammation was noted in all treated eyes one hour after treatment, at the 24 and 48-hour observations and in one treated eye at the 72-hour observation. Moderate to severe conjunctival irritation was noted in all treated eyes one hour after treatment with moderate conjunctival irritation at the 24 and 48-hour observations. Minimal to moderate conjunctival irritation was noted in all treated eyes at the 72-hour observation with minimal conjunctival irritation in one treated eye at the 7-day observation. All treated eyes appeared normal 7 - 14 days after treatment.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were equal to 1 for corneal opacity in all the animals tested, lower than 1 for iritis in 2 out of 3 animals, equal/higher than 2 for conjunctival redness in 2 out of 3 animals and lower than 2 for conjunctival oedema in 2 out of 3 animals. All treated eyes appeared normal 7 - 14 days after treatment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION

The study was performed to assess the irritancy potential of test substance following a single, 4-hour, semi-occluded application to the intact rabbit skin. The method used followed OECD guideline 404.

Faint yellow-coloured staining caused by the test material was noted at two treated skin sites during the study. It did not affect evaluation of skin responses.

Very slight erythema was noted at all treated skin sites 1, 24 and 48 hours after patch removal and continued to be noted at two treated skin sites at the 72-hour observation. Very slight oedema was noted at two treated skin sites one hour after patch removal. All treated skin sites appeared normal at the 7-day observation.

In addition, two old sheets reporting testing results are available. In both cases, details on testing procedures and results are lacking, thus reliability cannot be assigned. They are here mentioned only for completeness sake.

In one case, an irritation index was not calculated, in accordance with ETAD scoring system, and the substance is indicated to be not irritanting (sheet of test results, 1975). In the other case, an index of 1.5 is indicated, according to the ETAD scoring criteria, and the substance is indicated to be slightly irritating (sheet of test results, 1977).

EYE IRRITATION

The test item eye irritancy potential was investigated following OECD guideline 405. A volume of 0.1 ml of test material was placed into the conjunctival sac of the animal eye. Vocalisation was noted in one animal approximately ten seconds after dosing. Faint yellow-coloured staining caused by the test material was noted in all treated eyes during the study. This did not prevent accurate evaluation of ocular reactions. A dulling of the normal lustre of the corneal surface was noted in two treated eyes one hour after treatment. Areas of diffuse corneal opacity were noted in one treated eye one hour after treatment and in all treated eyes at the 24-hour observation. Diffuse corneal opacity persisted in all treated eyes up to the 72-hour observation and in one treated eye at the 7-day observation. Iridial inflammation was noted in all treated eyes one hour after treatment, at the 24 and 48-hour observations and in one treated eye at the 72-hour observation. Moderate to severe conjunctival irritation was noted in all treated eyes one hour after treatment with moderate conjunctival irritation at the 24 and 48-hour observations. Minimal to moderate conjunctival irritation was noted in all treated eyes at the 72-hour observation with minimal conjunctival irritation in one treated eye at the 7-day observation. All treated eyes appeared normal 7 - 14 days after treatment.

The mean values from gradings at 24, 48 and 72 hours were equal to 1 for corneal opacity in all animals tested, lower than 1 for iritis in 2 out of 3 animals, equal/higher than 2 for conjunctival redness in 2 out of 3 animals and lower than 2 for conjunctival oedema in 2 out of 3 animals. All treated eyes appeared normal 7 - 14 days after treatment.

A second experiment is available on the eye irritancy potential of Acid Yellow 218, using a test sample with low purity (ca. 47 % w/w a.i.). A volume of 0.1 g of the test material was placed into the rabbit's eyes. In the area of application a slight yellow discoloration of the sclera and conjunctivae was observed within 1 hour after treatment. This should be related to effects of the test article. No corrosion of the cornea was observed at each of the measuring intervals.

In this experiment, the mean values from gradings at 24, 48 and 72 hours were equal to 1 for corneal opacity in all the animals tested; the mean resulted to be lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema in all animals. All treated eyes appeared normal 7 days after treatment.

In both studies, treated eye were not rinsed during the tests. This fact might have increased the effect on the eye, however, following a precautionary approach, the substance is considered as eye irritant.

In addition, two old sheets reporting testing results are available. In both cases, details on testing procedures and results are lacking, thus a reliability cannot be assigned. They are here mentioned only for completeness sake.

In one case, an irritation index of 19 is reported, in accordance with ETAD scoring system, and the substance was indicated to be slightly irritant (sheet of test results, 1975). In the other case, severe redness and swelling lasting over 3 days are reported as effects; the substance is indicated to be mild irritating, according to the ETAD criteria (sheet of test results, 1977).

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), skin irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours. Mean value of ≥ 2,3 - ≤ 4,0 for erythema/eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal are taken as reference for classification as skin irritant.

The mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions. All treated skin sites appeared normal at the 7-day observation.

On these bases, the substance is not classified as skin irritant.

According to the CLP Regulation (EC 1272/2008), serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application. Eye irritation occurs if, when applied to the eye of an animal, a substance produces:

- at least in 2 of 3 tested animals, a positive response of: corneal opacity ≥ 1 and/or iritis ≥ 1 and/or conjunctival redness ≥ 2 and/or conjunctival oedema (chemosis) ≥ 2;

- calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

In the key study, the mean values from gradings at 24, 48 and 72 hours were equal to 1 for corneal opacity in all the animals tested, lower than 1 for iritis in 2 out of 3 animals, equal/higher than 2 for conjunctival redness in 2 out of 3 animals and lower than 2 for conjunctival oedema in 2 out of 3 animals. All treated eyes appeared normal 7 - 14 days after treatment.

Accordingly, the substance meets the criteria to be classified as eye irritant, category 2 (H319), according to the CLP Regulation (EC 1272/2008).