Amines, C36-alkylenedi-

Endpoint summary

Description of key information

Acute eye irritation/corrosion GLP study with C36-alkylenediamine according to OECD 405 showed irreversible effects on the eyes (Category 1).
In vivo dermal irritation/corrosion GLP study according to OECD 404 showed an extreme response following a 4-hour exposure to C36-alkylenediamine, which was irreversible within the 14-day observation period.

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:

04 - 18 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Animal used within the study was at least 6 weeks old
- Weight at study initiation: Body weight was at least 1.0 kg
- Housing: Animal was housed individually in cage with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad, Italy). Hay and wooden sticks were abailable during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Temporary deviations from the minimum level of daily mean relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 04 - 18 June 2012

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: Adjacent areas of the untreated skin of each animal served as controls.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL

Duration of treatment / exposure:

Single application.

Observation period:

14 days.

Number of animals:

one male

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and started with the treatment of one animal (sentinel) with a stepwise exposure regime. Based on the severity of the skin reactions, no further animals were exposed.

TEST SUBSTANCE PREPARATION
The test substance was applied undiluted as delivered by the sponsor.

TEST SITE
Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations.

The test substance was applied to the skin of one flank, using a Metalline patch of 2x3 cm.

REMOVAL OF TEST SUBSTANCE
Four hours after the application, the dressing was removed and the skin cleaned of residual test substance using water and ethanol.

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to application) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation: The skin reactions of all visible treated sites were assessed immediately after removal of a dressing and approximately 1, 24, 48, 72 hours after the removal of the last dressing and test substance. For the duration of the skin reactions, further observations were made 7 and 14 (maximum)
days after exposure. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of untreated skin of the animal served as controls.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system according to OECD 404.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours.

Score:

3

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours.

Score:

3

Max. score:

4

Irritant / corrosive response data:

Irritation: A 3-minute, 1-hour and 4-hour exposure resulted in erythema (maximum grade 4 (severe)) and oedema (maximum grade 2 (slight) to 4 (severe)), which had resolved within 14 days following exposure. Superficial brown discoloration (a sign of necrosis) and superficial fissuring of the skin were noted at 7 days after exposure, and bald skin (bald and shiny after a 1- and 4-hour exposure) and scaliness were noted at 14 days after exposure. At 7 days after a 1- and 4-hour exposure, skin reactions could not be scored due to fissuring and brown discolouration (a sign of necrosis; due to which a maximum grade 4 was given for erythema).

Corrosion: There was no evidence of a corrosive effect on the skin.

Other effects:

Coloration/remnants: On the skin exposed to the test substance for 1 and 4 hours, remnants of the test substance were present, being sticky on Day 1, and 24, 48 and 72 hours after exposure, and dry at 7 days after exposure. This did not hamper the scoring of the skin irritation reactions.

Toxicity/mortality: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

Category 2 (irritant)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

No evidence of full thickness destruction of the skin or scar tissue was observed during the observation period, indicating that no corrosion of the skin had occurred by dermal application of Amines, C36-alkylenedi- to the intact rabbit skin. The extreme response following a 4-hour exposure to Amines, C36-alkylenedi- in a single animal was irreversible within the 14-day observation period.
Based on these results and according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), Amines, C36-alkylenedi- should be classified as : skin irritant (Category 2).
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Amines, C36-alkylenedi- should be classified as Irritant (Category 2) and labeled as H315: Causes skin irritation.

Executive summary:

Primary skin irritation/corrosion study with AMINES, C36-ALKYLENEDI- in the rabbit (semi-occlusive application). The study was carried out based on the guidelines described in:

OECD No.404 (2002); "Acute Toxicity - Skin irritation"

EC, No 440/2008; B4: "Acute Toxicity: Dermal Irritation/Corrosion"

US EPA, OPPTS 870.2500 (1998), Acute Dermal Irritation

JMAFF Guidelines (2000) including the most recent partial revisions.

 

One rabbit was exposed to three samples of 0.5 mL of AMINES, C36-ALKYLENEDI- applied to separate skin-sites on intact, clipped skin using a semi-occlusive dressing. The exposure periods were 3 minutes, 1 hour and 4 hours, respectively. Skin reactions were assessed at least once daily for 4 days after exposure and 7 and 14 days after exposure. Based on the severity of the skin reactions, no further animals were exposed.

 

A 3-minute, 1-hour and 4-hour exposure resulted in erythema (maximum grade 4 (severe)) and oedema (maximum grade 2 (slight) to 4 (severe)), which had resolved within 14 days following exposure. Superficial brown discoloration (a sign of necrosis) and superficial fissuring of the skin were noted at 7 days after exposure, and bald skin (bald and shiny after a 1- and 4-hour exposure) and scaliness were noted at 14 days after exposure.

At 7 days after a 1- and 4-hour exposure, skin reactions could not be scored due to fissuring and brown discolouration (a sign of necrosis; due to which a maximum grade 4 was given for erythema).

There was no evidence of a corrosive effect on the skin.

 

On the skin exposed to the test substance for 1 and 4 hours, remnants of the test substance were present, being sticky on Day 1, and 24, 48 and 72 hours after exposure, and dry at 7 days after exposure. This did not hamper the scoring of the skin irritation reactions.

No evidence of full thickness destruction of the skin or scar tissue was observed during the observation period, indicating that no corrosion of the skin had occurred by dermal application of Amines, C36-alkylenedi- to the intact rabbit skin.

The extreme response following a 4-hour exposure to Amines, C36-alkylenedi- in a single animal was irreversible within the 14-day observation period.

 

Based on these results and according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), Amines, C36-alkylenedi- should be classified as : skin irritant (Category 2).

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Amines, C36-alkylenedi- should be classified as Irritant (Category 2) and labeled as H315: Causes skin irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

29 March - 19 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Animal used within the study was at least 6 weeks old
- Weight at study initiation: Body weights was at least 1.0 kg
- Housing: Animal was housed individually in cage with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 29 March - 19 April 2012

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied: 0.1 mL

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test substance

Number of animals or in vitro replicates:

1 male

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). Based on the nature and persistency of the ocular lesions observed during the study, the two further rabbits assigned to the study were not treated.

TREATMENT
On test Day 1, 0.1 mL of the test substance was instilled in the conjunctival sac of an eye of one animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of the animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. Immediately after fluorescein examination on Day 2, the treated eye was rinsed with approx 100 mL tepid tap water, using a velocity of flow which did not affect the eye, in an attempt to remove residual test substance. For reference control the other eye was also rinsed. After the final observation, the animal was sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
The in vivo test with one rabbit showed that the (test) substance present at 24 hours after instillation could only partly be removed from the eye by rinsing after 24 hours. Based on the results obtained for this animal, an ex-vivo test was conducted to assess the effectiveness of rinsing at 1 hour postinstillation (also in relation to worker exposure risk assessment). The severity of the eye lesions observed in the treated animal did not allow this test to be conducted in vivo in first instance.

REMOVAL OF TEST SUBSTANCE
-Washing: Yes, at 24 hours after instillation with 100 ml tepid tap water

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.The irritation was assessed according to OECD 405.

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

2

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Max. score:

2

Remarks on result:

other: Iris could not be scored due to (sticky) substance/secretions.

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

3

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

3

Max. score:

4

Irritant / corrosive response data:

Irritation: Instillation of 0.1 mL of Amines, C36-alkylenedi- into an eye of one rabbit resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity (maximum grade 3) and epithelial damage (maximum 65% of the corneal area). As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent at 7, 14 and 21 days after instillation. Between 1 hour and 14 days after instillation, only approximately 25% of the cornea could be scored due to presence of (sticky) substance/secretions. The iris showed no reaction to a light stimulus at 1 hour after instillation. Iridial irritation/responses could not be scored at 24, 48 and 72 hours, and 7 and 14 days after instillation due to presence of (sticky) substance/secretions. The irritation of the conjunctivae consisted of redness, chemosis and discharge. Redness remained present up to 21 days after instillation. Reduced flexibility of the eyelids was noted at 72 hours, and 7, 14 and 21 days after instillation, and a bald skin of the eyelids was noted at 7 and 14 days after instillation.

Corrosion: There was no evidence of ocular corrosion.

Other effects:

Colouration/Remnants: (Sticky) substance/secretions were noted on the cornea at 1, 24, 48 and 72 hours, and 7 and 14 days after instillation. Remnants of the test substance were present on the outside of the eyelids at 1, 24, 48 and 72 hours after instillation. The eyes were rinsed at 24 hours following instillation. An ex vivo test showed that the remnants could also not be removed from the cornea at 1 hour following instillation.

Toxicity/Mortality: No symptoms of systemic toxicity were observed in the animal during the test period and no mortality occurred.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results:
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), Amines, C36-alkylenedi- should be classified as : having irreversible effects on the eyes (Category 1).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Amines, C36-alkylenedi- should be classified as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.

Executive summary:

Acute eye irritation/corrosion study with Amines, C36-alkylenedi- was performed in the rabbit, according to OECD 405 and under GLP.

A single sample of 0.1 mL of Amines, C36-alkylenedi- was instilled into an eye of one rabbit. Observations were made 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation. Based on the severity and persistency of the eye lesions, no further animals were treated.

Instillation of the test substance resulted in effects on the cornea, iris and conjunctivae.

The corneal injury consisted of opacity (maximum grade 3) and epithelial damage (maximum 65% of the corneal area). As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent at 7, 14 and 21 days after instillation. Between 1 hour and 14 days after instillation, only approximately 25% of the cornea could be scored due to presence of (sticky) substance/secretions.

The iris showed no reaction to a light stimulus at 1 hour after instillation. Iridial irritation/responses could not be scored at 24, 48 and 72 hours, and 7 and 14 days after instillation due to presence of (sticky) substance/secretions. The irritation of the conjunctivae consisted of redness, chemosis and discharge. Redness remained present up to 21 days after instillation. Reduced flexibility of the eyelids was noted at 72 hours, and 7, 14 and 21 days after instillation, and a bald skin of the eyelids was noted at 7 and 14 days after instillation.

There was no evidence of ocular corrosion.

(Sticky) substance/secretions were noted on the cornea at 1, 24, 48 and 72 hours, and 7 and 14 days after instillation. Remnants of the test substance were present on the outside of the eyelids at 1, 24, 48 and 72 hours after instillation. The eyes were rinsed at 24 hours following instillation.

Anex vivotest showed that the remnants could also not be removed from the cornea at 1 hour following instillation.

 

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), Amines, C36-alkylenedi- should be classified as : having irreversible effects on the eyes (Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Eyes:

In first instance an in vitro screening for the eye irritancy potential of C36-alkylenediamine was performed using the Bovine Corneal Opacity and Permeability test (BCOP test).

The test substance was applied as it is (750 μl) directly on top of the corneas and left on place for 10±1 minutes. After the incubation the solutions were removed and the corneas washed with medium. Subsequently the corneas were incubated for 120±10 minutes at 32±1°C after which cornea opacity and permeability determination was performed.

Results from the negative and positive control substances were adequate, indicating that the test conditions were adequate and that the test system functioned properly.

In this test system, C36-alkylenediamine did not induce ocular irritation through both the endpoints opacity and permeability, resulting in a mean in vitroirritancy score of 5.7 after 10 minutes of treatment.

 

Subsequently an in vitro acute eye irritation/corrosion study was performed in the rabbit, according to OECD 405 and under GLP to confirm possible non-irritancy. A single sample of 0.1 mL of Amines, C36-alkylenedi- was instilled into an eye of one rabbit. Observations were made 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation. Based on the severity and persistency of the eye lesions, no further animals were treated.

(Sticky) substance/secretions were noted on the cornea at 1, 24, 48 and 72 hours, and 7 and 14 days after instillation. Remnants of the test substance were present on the outside of the eyelids at 1, 24, 48 and 72 hours after instillation. The eyes were rinsed at 24 hours following instillation.

An ex vivo test showed that the remnants could also not be removed from the cornea at 1 hour following instillation.

Instillation of the test substance resulted in effects on the cornea, iris and conjunctivae.

The corneal injury consisted of opacity (maximum grade 3) and epithelial damage (maximum 65% of the corneal area). As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent at 7, 14 and 21 days after instillation. Between 1 hour and 14 days after instillation, only approximately 25% of the cornea could be scored due to presence of (sticky) substance/secretions.

The iris showed no reaction to a light stimulus at 1 hour after instillation. Iridial irritation/responses could not be scored at 24, 48 and 72 hours, and 7 and 14 days after instillation due to presence of (sticky) substance/secretions.

The irritation of the conjunctivae consisted of redness, chemosis and discharge. Redness remained present up to 21 days after instillation. Reduced flexibility of the eyelids was noted at 72 hours, and 7, 14 and 21 days after instillation, and a bald skin of the eyelids was noted at 7 and 14 days after instillation.

There was no evidence of ocular corrosion.

Concluded was that the test substance C36-alkylenediamine should be classified as Category 1, irreversible effects on the eye.

 

Skin:

The in vitro BCOP study showed that the test substance was not a severe irritant or corrosive. However, the subsequent in vivo eye irritation study resulted to severe and irreversible effects on the eye. Previous experiences with various alkylamine derivatives in in vitro reconstructed human skin models have show that these tests severely underestimate the hazards for such substances as their highly corrosive hazards shown in vivo studies were not identified. As the in vivo eye irritation study showed no severe irritation of the eyelids it was considered justified to conduct an OECD 404 in vivo skin irritation study. By using the stepwise exposure regime, unnecessary animal harm is minimized to acceptable levels.

 

One rabbit was exposed to three samples of 0.5 mL of C36-alkylenediamine applied to separate skin-sites on intact, clipped skin using a semi-occlusive dressing and left for 3 minutes, 1 hour and 4 hours, respectively. Skin reactions were assessed at least once daily for 4 days after exposure and 7 and 14 days after exposure. Based on the severity of the skin reactions, no further animals were exposed.

A 3-minute, 1-hour and 4-hour exposure resulted in erythema (maximum grade 4 (severe)) and oedema (maximum grade 2 (slight) to 4 (severe)), which had resolved within 14 days following exposure. Superficial brown discoloration (a sign of necrosis) and superficial fissuring of the skin were noted at 7 days after exposure, and bald skin (bald and shiny after a 1- and 4-hour exposure) and scaliness were noted at 14 days after exposure.

At 7 days after a 1- and 4-hour exposure, skin reactions could not be scored due to fissuring and brown discolouration (a sign of necrosis; due to which a maximum grade 4 was given for erythema).

At the end of the 14-day observation period, all signs of oedema and erythema had disappeared, but still the skin displayed a bald, shiny, and scaliness aspect.

 

No evidence of full thickness destruction of the skin or scar tissue was observed during the observation period, indicating that no corrosion of the skin had occurred by dermal application of the test substance to the intact rabbit skin. The extreme response following a 4-hour exposure to Amines, C36-alkylenedi- in a single animal was irreversible within the 14-day observation period.

 

On the skin exposed to the test substance for 1 and 4 hours, remnants of the test substance were present, being sticky on Day 1, and 24, 48 and 72 hours after exposure, and dry at 7 days after exposure. This did not hamper the scoring of the skin irritation reactions. This stickiness leading to prolonged availability on the skin after the initial exposure duration could explain the comparable serious results from the 3 minutes and 1 hour exposures.

Based on these results, it was concluded that C36-alkylenediamine is extremely irritating to skin.

 

Respiratory:

No study is available. The substance is a very severe irritant. However, due to the very low vapour pressure (2.2 x10-5 hPa @ 20°C) inhalation of vapours leading to irritation of airways will not occur. Exposures to aerosols could possibly lead to respiratory irritation. Due to the low water solubility water based aerosols are not likely, and following the use mainly as industrial intermediate aerosol exposures are also not very likely.

Justification for selection of skin irritation / corrosion endpoint:
Only one study avaialble.

Justification for selection of eye irritation endpoint:
Only one adequate study available on in vivo results according to guidelines appropriate for classification.

Effects on skin irritation/corrosion: highly irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

An in vivo dermal irritation/corrosion GLP study according to OECD 404 showed an extreme response following a 4-hour exposure which was irreversible within the 14-day observation period. There was no evidence of a corrosive effect on the skin. Consequently, the substance C36-alkylenediamine should be classified as skin irritant (Category 2) and labeled as H315: Causes skin irritation.

An in vivo eye irritation/corrosion study in rabbit (OECD 405) resulted to effects on the cornea, iris and conjunctivae. The effects on cornea and conjunctivae were not resolved after 21 days. Although there was no evidence of ocular corrosion, the effects showed a corneal opacity of 3 for between days 7 and 14 (i.e. more than 3 consecutive days) and were with an opacity score of 2 still not recovered after 21 days. Also pannus and neovascularization of the cornea were observed at 21 days. These effects lead to classification the substance as Category 1, irreversible effects on the eye, with label H318: Causes serious eye damage.