N,N,N',N'-tetrakis(2,3-epoxypropyl)-m-xylene-α,α'-diamine

Endpoint summary

Description of key information

Skin irritation in vivo, a study similar to OECD404: irritating

Skin corrosion study in rat and mice (Japanese guideline): not corrosive

Eye irritation in vitro according to OECD438: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

5 Jun 1978 to 12 Jun 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

exposure duration of 24 hours instead of 4 hours/ occlusive coverage/ simultaneous testing on abraded skin

GLP compliance:

not specified

Specific details on test material used for the study:

Name cited in study report: PGA-X
Lot No: 80524
Appearance: colorless high viscous liquid

Species:

rabbit

Strain:

other: albino rabbits of Japanese native strain

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ichikawaya Limited
- Weight at study initiation: 2.6 to 3 kg
- Housing: The animals were housed individually in a conventional aluminum rabbit cage
- Diet: The animals were allowed free access to complete pelleted rabbit diet (Diet RC-4, Oriental Yeast Co.)
- Water: The animals were allowed free access to automatic watering.
- Acclimation period: 7 day observation period. During this period, all animals -were inspected for sigins of disease and skin condition

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 to 24
- Humidity (%): 50 to 60

Type of coverage:

occlusive

Preparation of test site:

clipped

Remarks:

application on intact and abraded skin

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0.5 mL neat substance

Duration of treatment / exposure:

24 hours

Observation period:

7 days

Number of animals:

six males

Details on study design:

TEST SITE
- Preparation of test site: Twenty-four hours before the intended application, the fur were clipped from the dorsal and blank regions of all animals by electric clippers. The abrasions were made by scratching across (2.5 x 2.5 cm) the stratum corneum of the epidermis by the point of a 23G needle.
- Type of wrap: 0.5 ml test compound was impregnated onto a surgical gauze patches measuring 1 by 1 inch and two single layer thick were applied on the abraded and intact skin of the rabbit. The patch was secured in place by 1.2 x 4 cm adhesive tape (Eratex). The entire trunk of the animal was wrapped with an impervious polystyrene film and covered with cloth for the 24-hours of exposure. The cloth material aids to maintain the test patches in position.

REMOVAL OF TEST SUBSTANCE
After 24 hours of exposure, the patches were removed and applied areas were rinsed with 200 mL tepid water. The plastic collar was fitted on the neck of the rabbit to prevent for the rabbit to access by mouth and to maintain the applied area during the observation period.

OBSERVATION TIME POINTS
Three hours after exposure, the first readings for the resulted reactions were made on the abraded and intact skin, and assigned to the 24 hour data point. The second reading were again done 74 hours after application. Scores were additionally determined at 48 hours and 7 days, these data were excluded for calculation of the overall score.

SCORING SYSTEM:
- Erythema and eschar formation and edema formation on applied areas were evaluated by the procedure described by Draize.
- The scores for erythema and eschar formation, for edema formation at 24 hours and at 72 hours after application on abraded skin were summed up. The total of four was divided by four to give the overall score for abraded skin. The overall score recorded for each animal was the averaged to give total average score for abraded skin. The total average score for intact skin was also given by the same way of calculations.

Irritation parameter:

erythema score

Basis:

animal: #1, #3, #6

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

erythema score

Basis:

animal: #4 and #5

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal: #1 and #5

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

edema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: intact skin

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: abraded skin

Irritation parameter:

edema score

Basis:

animal: #1, #2, #3 and #4

Time point:

24/48/72 h

Score:

3.7

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: abraded skin

Irritation parameter:

edema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: abraded skin

Irritation parameter:

edema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: abraded skin

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

other: 24 and 72 hour

Score:

4.08

Remarks on result:

other: intact skin

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

other: 24 and 72 hours

Score:

7.58

Remarks on result:

other: abraded skin

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

other: 24 and 72 hours

Score:

5.58

Remarks on result:

other: intact and abraded skin

Irritant / corrosive response data:

The negative control (water) did not cause erythema or edema in any of the animals on the intact skin. On the abraded skin, application of water resulted in an erythema score of 1 in all animals after 24 hours. In addition, an erythema score of 1 was assigned to 3 animals after 48 hours and to 1 animal after 72 hours.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this study the test item was considered to be irritating to the skin, based on the irritating effects seen on the intact skin.

Executive summary:

In a study, comparable to OECD404, 0.5 mL undiluted test substance was applied to the intact and abraded skin of six male albino rabbits of Japanese native strain under occlusive conditions. Animals were exposed for 24 hours after which the substance was removed with 200 mL tepid water. Application of water was included as negative control. Erythema and edema were scored according to the Draize scoring system 24, 48, 72 hours and 7 days after exposure. At the 24, 48 and 72 hour timepoints erythema scores of 2 or 3 were observed on the intact skin in all animals. The effects were, except for one animal, not fully reversible within 7 days. Edema scores were 1 (1 animal), 2 (2 animals) or 3 (3 animals) after 24 hours and 1 (3 animals) or 2 after (3 animals) after 48 hours. At the 72 hour the edema score was 0 or 1 (3 animals each). The edema was fully reversible in 7 days. Exposure on the abrades skin resulted in severe erythema and edema in all animals at all observed timepoints, and the effects were not (fully) reversible. Based on the effects observed on the intact skin, the substance has to be considered as irritating to the skin.   

Reference 2

Endpoint:

skin irritation / corrosion, other

Remarks:

corrosion study in mice

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: the test methods and evaluation criteria in the evaluation of danger of unknown substances established by the maritime techniques safety bureau, ministry of transport, Japan

Version / remarks:

December 1985

GLP compliance:

not specified

Specific details on test material used for the study:

Name as cited in study report: N,N,N',N'-tetraglycidyl metaxylenediamine (TETRAD-X)
Lot no.: 10K 22x
Appearance: pale brown transparent viscous liquid

Species:

mouse

Strain:

other: Crj-ICR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Co.
- Age at study initiation: 5 weeks
- Weight at study initiation: 29 to 31 g (male) and 24 to 26 g (female)
- Diet: Solid diet MF (Oriental Yeast Industries Co., Ltd) ad libitum
- Water: tap water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 6

Type of coverage:

open

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

1 mL/kg

Duration of treatment / exposure:

4 hours

Observation period:

4 hours

Number of animals:

3 males and 3 females

Details on study design:

TEST SITE AND EXPOSURE
- Each animal was fixed in the prone position on a fixing board; the skin in the back was exposed as the hair was clipped using an electric clipper for small animals.
- The animal was left for about 2 hours, and then was determined to have neither injuries nor rash due to hair clip.
- After that, the exposed skin was thoroughly cleaned using gauze wetted with water and the substance was applied.
- Area of exposure: 1cm2/ head
- Type of wrap: No covering

OBSERVATION TIME POINTS
- For the evaluation of severities of erythema, edema, haemorrhage and necrosis, changes appearing in the skin at 3 minutes, 60 minutes and 4 hours after application of the test substance were macroscopically observed while the animals stood in fixation.
- At the termination of the 4-hour observation, the skin was incised, and then the cut surfaces and subcutaneous region in the applied site were examined.

SCORING SYSTEM:
Corrosiveness of the test substance was evaluated based on dermal changes appearing at 3 minutes, 60 minutes and 4 hours after application in accordance with the criteria established by the International Maritime Organization (see any other information on materials and methods incl. tables). If one of the 6 animals developed necrosis in the skin within 4 hours after application, the test substance must be judged to be a corrosive substance.

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

other: 3 min and 60 min

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #4, #5 and #6

Time point:

other: 4h

Score:

1

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 4h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

other: 3 min and 60 min

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #1, #3 and #5

Time point:

other: 4h

Score:

3

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #2, #4 and #6

Time point:

other: 4h

Score:

2

Max. score:

4

Irritant / corrosive response data:

Although there were no changes until 60 minutes after application, a large number of moderate edemas appeared in the nodular form after 4 hours in addition to mild erythema; edematous sites were red in color in female mice. However, none of the animals showed macroscopic changes judged to be necrosis. In the observation of cut surfaces of the skin at 4 hours after application, small prominent and nodular changes were determined to be edemas; a small amount of liquid retention in the subcutaneous tissue was noted in female mice. Neither necrosis nor subcutaneous haemorrhage was observed.

Interpretation of results:

other: not corrosive

Remarks:

in accordance with CLP (1272/2008 and its updates)

Conclusions:

Although marked irritative changes with edema were observed at 4 hours after application, the substance is not considered to be corrosive.

Executive summary:

An in vivo skin corrosion test was performed in mice. In this test 1 mL/kg test substance was applied on the intact skin on the back three male and three female Crj-ICR mice for an exposure period of 4 hours. For the evaluation of severities of erythema, edema, hemorrhage and necrosis, changes appearing in the skin at 3 minutes, 60 minutes and 4 hours after application of the test substance were macroscopically observed while the animals stood in fixation. At the termination of the 4-hour observation, the skin was incised, and then the cut surfaces and subcutaneous region in the applied site were examined. Although there were no changes until 60 minutes after application, a large number of moderate edemas appeared in the nodular form after 4 hours in addition to mild erythema; edematous sites were red in colour in female mice. However, none of the animals showed macroscopic changes judged to be necrosis. In the observation of cut surfaces of the skin at 4 hours after application, small prominent and nodular changes were determined to be edemas; a small amount of liquid retention in the subcutaneous tissue was noted in female mice. Neither necrosis nor subcutaneous haemorrhage was observed. Based on the observed effects, the substance was not considered to be corrosive.

Reference 3

Endpoint:

skin irritation / corrosion, other

Remarks:

corrosion study in rat

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: the test methods and evaluation criteria in the evaluation of danger of unknown substances established by the maritime techniques safety bureau, ministry of transport, Japan

Version / remarks:

December 1985

GLP compliance:

not specified

Specific details on test material used for the study:

Name as cited in study report: N,N,N',N'-tetraglycidyl metaxylenediamine (TETRAD-X)
Lot no.: 10K 22x
Appearance: pale brown transparent viscous liquid

Species:

rat

Strain:

other: Crj-Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Co.
- Age at study initiation: 5 weeks
- Weight at study initiation: 231 to 238 g (male) and 146 to 160 g (female)
- Diet: Solid diet MF (Oriental Yeast Industries Co., Ltd) ad libitum
- Water: tap water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 6

Type of coverage:

open

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

1 mL/kg

Duration of treatment / exposure:

2 hours

Observation period:

4 hours

Number of animals:

3 males and 3 females

Details on study design:

TEST SITE AND EXPOSURE
- Each animal was fixed in the prone position on a fixing board; the skin in the back was exposed as the hair was cropped using an electric clipper for small animals.
- The animal was left for about 2 hours, and then was determined to have neither injuries nor rash due to hair clip.
- After that, the exposed skin was thoroughly cleaned using gauze wetted with water and the substance was applied.
- Area of exposure: 1cm2/ head
- Type of wrap: No covering

OBSERVATION TIME POINTS
- For the evaluation of severities of erythema, edema, hemorrhage and necrosis, changes appearing in the skin at 3 minutes, 60 minutes and 4 hours after application of the test substance were macroscopically observed while the animals stood in fixation.
- At the termination of the 4-hour observation, the skin was incised, and then the cut surfaces and subcutaneous region in the applied site were examined

SCORING SYSTEM:
Corrosiveness of the test substance was evaluated based on dermal changes appearing at 3 minutes, 60 minutes and 4 hours after application in accordance with the criteria established by the International Maritime Organization (see any other information on materials and methods incl. tables). If one of the 6 animals developed necrosis in the skin within 4 hours after application, the test substance must be judged to be a corrosive substance.

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

other: 3 min

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: #1, #3 and #5

Time point:

other: 60 min

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: #2, #4 and #6

Time point:

other: 60 min

Score:

1

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #4, #5 and #6

Time point:

other: 4h

Score:

1

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 4h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #1, #2, #3, #4, #5 and #6

Time point:

other: 3 min and 60 min

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal: #1, #3, #4, #5 and #6

Time point:

other: 4h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 4h

Score:

2

Max. score:

4

Irritant / corrosive response data:

Changes in rats were mild; erythema appeared after 60 minutes, and small prominent edemas were sporadically seen at a very small degree after 4 hours in addition to erythema. Neither necrosis nor subcutaneous haemorrhage was observed.

Interpretation of results:

other: not corrosive

Remarks:

in accordance with CLP (1272/2008 and its updates)

Conclusions:

Signs of mild irritation were observed 4h after application. Therefore the substance was not considered to be corrosive.

Executive summary:

An in vivo skin corrosion test was performed in rats. In this test 1 mL/kg test substance was applied on the intact skin on the back three male and three female Crj-Wistar for an exposure period of 4 hours. For the evaluation of severities of erythema, edema, hemorrhage and necrosis, changes appearing in the skin at 3 minutes, 60 minutes and 4 hours after application of the test substance were macroscopically observed while the animals stood in fixation. At the termination of the 4-hour observation, the skin was incised, and then the cut surfaces and subcutaneous region in the applied site were examined. Changes rats were mild; erythema appeared after 60 minutes, and small prominent edemas were sporadically seen at a very small degree after 4 hours in addition to erythema. Neither necrosis nor subcutaneous haemorrhage was observed. Based on the observed effects, the substance was not considered to be corrosive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

02 Aug 2017 to 27 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist

Specific details on test material used for the study:

Batch no.: 70518-X
Purity: N.A.
Expiry date: 18/08/2017
Appearance: yellowish liquid

Species:

other: Eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL neat substance

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Test group and positive control: triplicates
Negative control: Singlo

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Other effects / acceptance of results:

Slit-lamp examination: The test substance caused corneal effects consisting of very slight or slight opacity (mean score of 0.7) and no or very slight fluorescein retention (mean score of 0.2). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
Microscopic examination of the revealed very slight erosion (2/3 corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% generally revealed severe erosion and very slight vacuolation (mid/low regions) of the epithelium, pyknotic nuclei in the stroma, and endothelial necrosis.

Interpretation of results:

other: Not irritating

Remarks:

Based on CLP criteria (EC 1272/2008 and its updates)

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is considered not considered to be an eye irritant in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).

Executive summary:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5% Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused corneal effects consisting of very slight or slight opacity (mean score of 0.7) and no or very slight fluorescein retention (mean score of 0.2). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the revealed very slight erosion (2/3 corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% generally revealed severe erosion and very slight vacuolation (mid/low regions) of the epithelium, pyknotic nuclei in the stroma, and endothelial necrosis. Based on these results, the substance is not considered to be irritating to the eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vivo (similar to OECD404)

In a study, comparable to OECD404, 0.5 mL undiluted test substance was applied to the intact and abraded skin of six male albino rabbits of Japanese native strain under occlusive conditions. Animals were exposed for 24 hours after which the substance was removed with 200 mL tepid water. Application of water was included as negative control. Erythema and edema were scored according to the Draize scoring system 24, 48, 72 hours and 7 days after exposure. The average of the 24, 48 and 72 hour timepoints for erythema scores were between 2 and 3 when applied on the intact skin in all animals. The effects were, except for one animal, not fully reversible within 7 days. Average edema scores were between 0.7 and 2 and was fully reversible in 7 days. Exposure on the abrades skin resulted in severe erythema and edema in all animals at all observed timepoints, and the effects were not (fully) reversible. Based on the effects observed on the intact skin, the substance has to be considered as irritating to the skin (Mites 1978).   

Skin irritation in vivo (similar to OECD404 with diluted substance)

In a study, comparable to OECD404, 0.15 mL of five different test substance dilutions were applied on distinct regions of the intact skin of six male albino rabbits of Japanese native strain under occlusive conditions. The applied concentrations were 0.1, 0.4, 1.6, 6.4 and 25.6% dissolved in acetone. A vehicle control was taken along. Animals were exposed for 24 hours after which the substance was removed with 200 mL tepid water. Application of the vehicle was included as control. Erythema and edema were scored according to the Draize scoring system 24, 48, 72 hours and 7 days after exposure. Irritating effects were observed mostly at the sites treated with 6.4 and 25.6% test substance. Erythema scores of 1 to 3 were obtained in in the 6.4% dose group after 24 hours, but the effects were reversible within 7 days, except for one animal. Edema was scored between 0 to 2 after 24 hours and was fully reversible. After application of 25.6% the erythema score was 2 to 3 after 24 and 48 hours and 1 or 2 after 72 hours. The effects were fully reversible within 7 days in half of the animals. The edema score were 0 or 1 and fully reversible in the 7 day observation period. For one animal an edema score of 2 was obtained at the 24 and 48 timepoints, these effects were reversible as well. Based on the effects observed on the intact skin, the diluted substance at 25.6% has to be considered as irritating to the skin (Mites 1978).   

Skin corrosion study in mice

An in vivo skin corrosion test was performed in mice. In this test 1 mL/kg test substance was applied on the intact skin on the back three male and three female Crj-ICR mice for an exposure period of 4 hours. For the evaluation of severities of erythema, edema, hemorrhage and necrosis, changes appearing in the skin at 3 minutes, 60 minutes and 4 hours after application of the test substance were macroscopically observed while the animals stood in fixation. At the termination of the 4-hour observation, the skin was incised, and then the cut surfaces and subcutaneous region in the applied site were examined. Although there were no changes until 60 minutes after application, a large number of moderate edemas appeared in the nodular form after 4 hours in addition to mild erythema; edematous sites were red in colour in female mice. However, none of the animals showed macroscopic changes judged to be necrosis. In the observation of cut surfaces of the skin at 4 hours after application, small prominent and nodular changes were determined to be edemas; a small amount of liquid retention in the subcutaneous tissue was noted in female mice. Neither necrosis nor subcutaneous haemorrhage was observed. Based on the observed effects, the substance was not considered to be corrosive (Niigata 1986).

Skin corrosion study in rat

An in vivo skin corrosion test was performed in rats. In this test 1 mL/kg test substance was applied on the intact skin on the back three male and three female Crj-Wistar for an exposure period of 4 hours. For the evaluation of severities of erythema, edema, hemorrhage and necrosis, changes appearing in the skin at 3 minutes, 60 minutes and 4 hours after application of the test substance were macroscopically observed while the animals stood in fixation. At the termination of the 4-hour observation, the skin was incised, and then the cut surfaces and subcutaneous region in the applied site were examined. Changes rats were mild; erythema appeared after 60 minutes, and small prominent edemas were sporadically seen at a very small degree after 4 hours in addition to erythema. Neither necrosis nor subcutaneous haemorrhage was observed. Based on the observed effects, the substance was not considered to be corrosive (Niigata 1986).

Eye irritation in vitro (OECD438)

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5% Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused corneal effects consisting of very slight or slight opacity (mean score of 0.7) and no or very slight fluorescein retention (mean score of 0.2). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the revealed very slight erosion (2/3 corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% generally revealed severe erosion and very slight vacuolation (mid/low regions) of the epithelium, pyknotic nuclei in the stroma, and endothelial necrosis. Based on these results, the substance is not considered to be irritating to the eyes (Triskelion 2017).

Justification for classification or non-classification

According to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 classification of the substance for eye irritation is not warranted.

According to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 the substance has to be classified as a skin irritant Category 2.