Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Endpoint summary

Description of key information

Skin irritation, in vivo EPA OPPTS 870.2500 study; not classified

Eye irritation, in vivo OECD 405 study; not classified

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:

Initiated 11Sep2001; Reported 19Oct2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

The test article, MIP 2982, Lot Number 028400A8AA (Vibracolor Yellow) (identified at receipt as MIP Yellow 2982, Batch Number 028400A8AA, Expiration Date: September 2006), was received fix)m the Sponsor on September 10,2001. The test article was received and stored under ambient conditions. Upon receipt, the test article was described as yellowish-orange powder with chunks.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test Animals
Naive, young-adult, albino Hra:(NZW)SPF rabbits were received on September 06, 2001 from Covance Research Products, Inc., a USDA-approved supplier located in Denver, Pennsylvania.

Housing
The animals were housed individually in stainless steel, screen-bottomed cages, measuring 60.7 cm X 60.3 cm x 40.7 cm (DxWxH).

Diet
The animals were presented with a measured amount of PMI* Feeds Certified Rabbit High Fiber Diet #5325 daily. Each lot of feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients.

Water
Water was available ad libitum throughout the acclimation and study periods. Samples of the water were analyzed by a certified laboratory approximately every 6 months for specified microorganisms and environmental contaminants.

Nutrient and Contaminant Analyses
Specified nutrient and contaminant analyses are on file at Covance - Vienna. There were no contaminants, known or reasonably anticipated, in the diet or water at levels that might interfere with the validity of this study.

Environment
Controls in the animal room were set to maintain temperatures and relative humidity of 16-22°C (61-72°F) and 50±20%, respectively, a 12-hour light/12-hour dark cycle (lights on approximately 0600 to 1800 hours), and 10 or greater air changes per hour.

Acclimation
Before being considered for study use, the animals were acclimated to laboratory conditions. After at least 5 days of acclimation, a staff veterinarian deemed them to be healthy and free from disease and physical abnormalities, and then released the animals for use in the study.

Environmental Enrichment
The animals were given plastic balls as a form of environmental enrichment.

Selection of Animals
Only healthy animals, free from skin defects or alterations in coloration or texture, were used in this study. Three rabbits (two males and one female), approximately 14 weeks old and weighing from 2328 to 2694 g, were randomly selected from the animals available for this study. Each animal was assigned a unique animal number and affixed with an ear tag marked with the corresponding animal number; the corresponding number was also affixed to the cage housing the animal.

Justification of Species Selection
Historically, the New Zealand White albino rabbit has been the animal of choice for evaluating the effects of chemicals on the skin. This species is designated by federal regulations and has a large historical database for dermal irritation assays.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Remarks:

0.5 mL distilled water

Amount / concentration applied:

Approximately 0.5 g of test material in a paste with 0.5mL distilled water

Duration of treatment / exposure:

4-hour exposure period

Observation period:

Dermal irritation was evaluated at approximately 0.5 to 1, 24,48, and 72 hours after patch removal.

Number of animals:

Two males and one female

Details on study design:

Dose Administration
On the day of dosing, the test material was finely ground with a mortar and pestle and pasted with approximately 0.5 mL distilled water (Crystal Springs, Lot No. BTLD:03/20/01 LANP Exp:03/20/03) prior to application. A total of 0.5 g of the test article was applied to one intact test area (approximately 6.25 cm^) of each animal. To provide a semi-occlusive dressing, each area of exposure was covered with a gauze patch secured with paper tape, loosely overwrapped with Saran Wrap , and secured with Elastoplast tape. The animals were uncollared during the 4-hour exposure period. The dressing for each animal remained intact during the 4-hour exposure period. The adjacent, untreated skin sites of each animal served as the control.

Removal of Test Article
After a 4-hour exposure period, the dressings were removed. The residual test article was removed by spraying the treated area with tap water and soap and gently wiping with soft paper towels.

Body Weights
The animals were weighed prior to test article application.

Health and Mortality
The animals were observed twice daily (at least 4 hours apart) for general health and mortality for the duration of the study.

Reading of Dermal Irritation
Approximately 0.5 to 1,24,48, and 72 hours after patch removal, the exposure site was examined for the degree of erythema (redness) and edema (swelling). The adjacent, untreated clipped area on each animal's back was used for control. The test sites were scored according to the primary dermal irritation scoring scale (see report for details).

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no irritation observed

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Other effects:

Animals did not exhibit gross evidence of treatment-related toxicity during the course of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

No findings of erythema or edema were noted in any animal at any observation period. The Primary Dermal Irritation Index (PDII) was calculated to be 0.0. No evidence of corrosion was observed. The test article is considered to be non-irritating to the skin of the rabbit under the conditions of this study.

Executive summary:

The acute dermal irritation of the test article on rabbits under semi-occluded conditions was evaluated in general compliance with the conditions specified in the US EPA Health Effects Testing Guidelines OPPTS 870.2500 Acute Dermal Irritation (dated August 1998) and the OECD Guidelines for Testing of Chemicals, Section 4, Health Effect, No 404.

 

On the day of dosing, approximately 0.5 g of the test article was applied to one intact test area (approximately 6.25 cm2) on each back of three New Zealand White rabbits (two males and one female) for a 4-hour exposure period. Dermal irritation was evaluated at approximately 0.5 to 1, 24,48, and 72 hours after patch removal.

 

Animals did not exhibit gross evidence of treatment-related toxicity during the course of the study.

 

No findings of erythema or edema were noted in any animal at any observation period. The Primary Dermal Irritation Index (PDII) was calculated to be 0.0. No evidence of corrosion was observed. The test article is considered to be non-irritating to the skin of the rabbit under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06Feb2015 to 10 Mar2015

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)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material name : Vibracolor® Citrus Yellow
Chemical name : Methyl -1-methyl-4-((methylphenylhydrazono)
methyl)pyridinium sulphate
Batch number : 0010241302
BASF Test Item no.
(Container identification) : 10/0384-4
Purity : 99.2% (see Annex 3)
Appearance : solid, yellow
Storage conditions : ambient temperature (15-25ºC)
Expiry date : 21/06/2018

Species:

chicken

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

Characterization of the test system
Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5 - 2.5 kg, were used as eye-donors. Heads of these animals were obtained from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands. 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.

Experimental design
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 BP 2.0% w/v (Minims, Chauvin, England) 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 ndamaged (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 (TNO Triskelion, Zeist, the Netherlands). 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 (peristaltic pump set at speed 5.00, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32oC (water pump set at 36.4oC; Lauda 103, Germany).

After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope. Corneal thickness was expressed in instrument units. 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.
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.

Vehicle:

other: Either directly (30 mg) or as 5% aqueous solution (30 μL)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

Negative control; Physiological saline; 30 μL
Positive control; NaOH; 30 mg
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 30 mg
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 30 μL

Duration of treatment / exposure:

Negative control; Physiological saline; 10 seconds exposure then washed with 20mL saline
Positive control; NaOH; 10 seconds exposure then washed with 20mL saline
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 10 seconds exposure then washed with 20mL saline
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 10 seconds exposure then washed with 20mL saline

Duration of post- treatment incubation (in vitro):

After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment

Number of animals or in vitro replicates:

Negative control; Physiological saline; 1 eye
Positive control; NaOH; 3 eyes
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 3 eyes
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 3 eyes

Details on study design:

Negative control; Physiological saline; 10 seconds exposure then washed with 20mL saline
Positive control; NaOH; 10 seconds exposure then washed with 20mL saline
Test group 1; Vibracolor® Citrus Yellow (no vehicle); 10 seconds exposure then washed with 20mL saline
Test group 2;Vibracolor® Citrus Yellow - 5% aqueous; 10 seconds exposure then washed with 20mL saline

After rinsing, each eye in the holder was returned to its chamber.
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment.
Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
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.

Irritation parameter:

percent corneal swelling

Run / experiment:

Vibracolor Citrus Yellow 30 mg

Value:

4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Vibracolor Citrus Yellow 30 mg

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

Vibracolor Citrus Yellow 30 mg

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Slit-lamp examination
An overall summary of the results of the control (negative and positive) and test substances based on maximum mean values for corneal swelling, corneal opacity, and fluorescein retention, the irritation categories assigned to the parameters. The individual and mean values for corneal swelling, corneal opacity and fluorescein retention are recorded in the study records.

Vibracolor® Citrus Yellow caused corneal effects consisting of very slight corneal swelling (mean of 4%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). The opacity and fluorescein retention were observed as spots. In addition, yellow staining of cornea and sclera was observed which persisted until the end of the observation period.

The 5% aqueous dilution of Vibracolor® Citrus Yellow only caused very slight corneal swelling (mean of 1%). The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate

The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination
Since the test sample concerned a dye substance, the vitreous body of each eye was examined for possible staining during collection of the corneas in formalin and no staining was observed.

Microscopic examination of the corneas treated with Vibracolor® Citrus Yellow revealed very slight erosion of the epithelium in two corneas. Microscopic examination of the corneas treated with a 5% aqueous dilution of Vibracolor® Citrus Yellow did not reveal any abnormalities.

Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, pyknotic nuclei in the inner region of the stroma and necrosis of the endothelium.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Applying the classification criteria of the ICE, the following irritation classifications can be assigned to the test item:
- Category 2A:“Irritant/causes eye irritation” (UN-GHS classification);
- Category 2:“Irritating to eyes” (EU-CLP classification).

5% aqueous dilution of the test item:
- Not Classified (UN-GHS and EU-CLP classifications).

Executive summary:

Test item and a 5% aqueous dilution of the test item were evaluated for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 μL (liquids) or 30 mg (solids) for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

 

Vibracolor® Citrus Yellow caused corneal effects consisting of very slight corneal swelling (mean of 4%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). The opacity and fluorescein retention were observed as spots. In addition, yellow staining of cornea and sclera was observed which persisted until the end of the observation period. The 5% aqueous dilution of the test item only caused very slight corneal swelling (mean of 1%).

 

Since the test sample concerned a dye substance, the vitreous body of each eye was examined for possible staining during collection of the corneas in formalin and no staining was observed. Microscopic examination of the corneas treated with the test item revealed very slight erosion of the epithelium in two corneas. Microscopic examination of the corneas treated with a 5% aqueous dilution of the test item did not reveal any abnormalities.

 

The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, pyknotic nuclei in the inner region of the stroma and necrosis of the endothelium.

 

Applying the classification criteria of the ICE, the following irritation classifications can be assigned to the test item:

-Category 2A:“Irritant/causes eye irritation” (UN-GHS classification);

-Category 2:“Irritating to eyes” (EU-CLP classification).

5% aqueous dilution of the test item:

-Not Classified (UN-GHS and EU-CLP classifications).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05Sep2001 to 21Jan2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

The test article, MIP 2982 (Lot No. 028400A8AA, Vibracolor yellow), was received from the Sponsor on September 10, 2001. The test article (identified as MIP Yellow 2982 upon receipt) was received and stored at ambient temperature. Upon receipt, the test article was described as a yellowish-orange powder with chunks.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animals
Naive, young-adult, albino Hra:(NZW)SPF rabbits were received on October 30, 2001 from Covance Research Products, Inc., a USDA-approved supplier located in Denver, Pennsylvania.

Housing
The animals were housed individually in stainless-steel, screen-bottomed cages, measuring 60.7 cm X 60.3 cm x 40.7 cm (DxWxH).

Diet
The animals were presented with a measured amount of PMI® Feeds Certified Rabbit High Fiber Diet #5325 daily. Each lot of feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified
nutrients.
Water
Water was available ad libitum throughout the acclimation and study periods. Samples of the water were analyzed by a certified laboratory approximately every 6 months for specified microorganisms and environmental contaminants.

Nutrient and Contaminant Analyses
Specified nutrient and contaminant analyses are on file at Covance - Vienna. There were no contaminants, known or reasonably anticipated, in the diet or water at levels that might interfere with the validity of this study.

Environment
Controls in the animal room were set to maintain temperatures and relative humidity of 16-22°C (61-72°F) and 50±20%, respectively, a 12-hour light/12-hour dark cycle (lights on approximately 0600 to 1800 hours), and 10 or greater air changes per hour.

Acclimation
Before being considered for study use, the animals were acclimated to laboratory conditions. After at least 5 days of acclimation, a staff veterinarian deemed them to be healthy and free from disease and physical abnormalities, and then released the animals for use in the study.

Environmental Enrichment
The animals were given plastic balls as a form of environmental enrichment.

Selection of Animals
Three, naive, young-adult rabbits (one male and two females), approximately 13 weeks old and weighing from 2216 to 2477 g, were randomly selected from the animals available for this study. Each animal was assigned a unique animal number and affixed with an eartag marked with the corresponding animal number; the corresponding number was also affixed to the cage housing
the animal.

Justification of Species Selection
Historically, the New Zealand White albino rabbit has been the animal of choice for evaluating the effects of chemicals on the eyes because of its large orbit and nonpigmented iris. This species is designated by federal regulations and has a large historical database for ocular irritation assays.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

approximately 0.057 g per test eye

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

Approximately I, 24,48, 72, and 96 hours and 7, 14, and 21 days after instillation of the test article, the treated eyes of the rabbits were observed for ocular irritation.

Number of animals or in vitro replicates:

3 animals (2 Female, 1 Male)

Details on study design:

Approximately 24 hours after instillation of the test article, the treated eyes of the animals were examined for residual test article; any residual test article (if any) was gently washed out using room-temperature physiological saline.
Approximately I, 24,48, 72, and 96 hours and 7, 14, and 21 days after instillation of the test article, the treated eyes of the rabbits were observed for ocular irritation. The contralateral untreated eye was used as the control. Corneal injury was assessed using sodium fluorescein dye (followed by a saline wash) on all animals at 24 hours post instillation. Because there was no injury observed at this interval, no further assessment using sodium fluorescein dye was performed. Irritation was graded as noted in the report, per Draize Technique.

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

other: 1 hour

Score:

ca. 9

Max. score:

9

Reversibility:

other: Iris findings resolved within 24 hours. Redness persisted in one animal up to day 21. Chemosis was resolved by 72 hours.

Remarks on result:

other: Findings at 1 hour are not included in the determination of classification

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: 1 hour

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Irritation criteria not met

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effect

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Irritation criteria not met

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Irritation criteria not met

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks:

Spurious and Inconclusive finding on reversibility as scored 0 at 96 h and 7 day giving evidence of reversibility. The score of 1 on day 14 and 21 is spurious.

Remarks on result:

other: Irritation criteria not met

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Irritation criteria not met

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Irritation criteria not met

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: Irritation criteria not met

Irritant / corrosive response data:

There was no indication of pain in any animal upon instillation of the test article or shortly thereafter. Animals did not exhibit gross evidence of treatment-related toxicity during the course of the study.

There were no findings involving the cornea. Findings involving the iris were limited to circumcomeal injection with the iris still reacting to light (Score 1) in one animal and slow reaction to light (Score 0) in a second animal at 1 hour postinstillation. These findings were resolved by 24 hours postinstillation.Note findings at 1 hour post installation are not used for the pupose of classification.

Redness (Score 1 or 2) in all animals from 1 to 72 hours postinstillation, in two animals at 96 hours postinstillation, and in one animal/timepoint 7, 14, and 21 days postinstillation. Chemosis (Score 1 or 2) was noted in all animals at 1 and 24 hours postinstillation and in two animals at 48 hours postinstillation. Discharge (Score 1 or 2) was noted in one animal at 1 hour postinstillation, in three animals at 24 hours postinstillation, and in two animals at 48 hours postinstillation. There was no evidence of corrosion noted in any animal during the course of the study. Note findings at 1 hour post installation are not used for the pupose of classification.

According to Regulation (EC) 1272/2008 Eye Irritation Category 2 is based on:

Substances that produce in at least in 2 of 3 tested animals, a positive response of:

(a) corneal opacity ≥ 1 and/or

(b) iritis ≥ 1, and/or

(c) conjunctival redness ≥ 2 and/or

(d) 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

Cornea - scores were 0 at all time points.

Iris: A single score of 1 was recored for animal #3 at 1 hour post installation. All other scores were 0.

Conjunctivae: the mean scores over 24, 48 and 72 hours in this study are shown below:

	conjunctivae
	redness			chemosis
time	6279	6280	6281	6279	6280	6281
24	1	1	1	1	2	1
48	2	1	1	1	1	0
72	1	1	1	0	0	0
mean	1.33	1.00	1.00	0.67	1.00	0.33

(A EURL ECVAM excel sheet used to calculate means of 24/48 and 72 hours has been attached)

When the mean grading of 24, 48 and 72 hours are taken into consideration the criteria for Irritation Category 2 are not met. When reversibility is taken into consideration the findings in animals #1 and #2 are fully reversible. For Animal #3, reversibility was demonstrated with a score of 0 on 2 consecutive occasions

(96 hours and 7 days). Following this a score of 1 was reported on day 14 and 21; this was therefore considered to be spurious. It may therefore be concluded that the findings were reversible.

Interpretation of results:

GHS criteria not met

Conclusions:

From the findings in this study and according to the criteria described in Regulation (EC) 1272/2008, where in at least 2 of 3 tested animals, there are findings of conjunctival redness >= 2 and/or Conjunctival oedema >=2 a substance fulfills the criteria for the classification of Irritating to eyes, category 2.
On this basis the test substance mean scores at 24, 48 and 72 hours were less than 1 for corneal opacity and iritis and less than 2 for conjunctival redness or chemosis, and therefore it was considered that the criteria for classification were not met.

Executive summary:

This 2002 in vivo primary eye irritation study was performed acording to the US EPA Health Effects Test Guidelines OPPTS 870.2400 Acute Eye Irritation (dated August, 1998) and the OECD Guidelines for Testing of Chemicals, Section 4, Health Effect, No. 405.

The test substance was applied by the installation of 0.057 g of the powdered test substance (with any chunks broken down with use of a spatula) into the everted lower lid of the right eye and then both lids were gently held together for 1 second. The left eye was used as an untreated control.

Readings of ocular irritation were performed at 1, 24, 48, 72, 96 hours after application and again at 7, 14 and 21 days.

Findings in the eyes were graded and scored according to the method of Draize.

Note the readings at 1 hour were not used in the criteria for classification according to Regulation (EC) 1272/2008.

According to Regulation (EC) 1272/2008 Eye Irritation Category 2 is based on:

Substances that produce in at least in 2 of 3 tested animals, a positive response of:

(a) corneal opacity ≥ 1 and/or

(b) iritis ≥ 1, and/or

(c) conjunctival redness ≥ 2 and/or

(d) 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

Cornea - scores were 0 at all time points.

Iris: A single score of 1 was recored for animal #3 at 1 hour post installation. All other scores were 0.

Conjunctivae: the mean scores over 24, 48 and 72 hours in this study are shown below:

	conjunctivae
	redness			chemosis
time	6279	6280	6281	6279	6280	6281
24	1	1	1	1	2	1
48	2	1	1	1	1	0
72	1	1	1	0	0	0
mean	1.33	1.00	1.00	0.67	1.00	0.33

When the mean grading of 24, 48 and 72 hours are taken into consideration the criteria for Irritation Category 2 are not met. When reversibility is taken into consideration the findings in animals #1 and #2 are fully reversible. For Animal #3, reversibility was demonstrated with a score of 0 on 2 consecutive occasions

(96 hours and 7 days). Following this a score of 1 was reported on day 14 and 21; this was therefore considered to be spurious. It may therefore be concluded that the findings were reversible.

From the findings in this study and according to the criteria described in Regulation (EC) 1272/2008, the criteria for classification were not met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

An in vivo and an in vitro study are available for the investigation of eye irritation.

A 2002 in vivo primary eye irritation study was performed acording to the US EPA Health Effects Test Guidelines OPPTS 870.2400 Acute Eye Irritation (dated August, 1998) and the OECD Guidelines for Testing of Chemicals, Section 4, Health Effect, No. 405. The study was allocated a reliability score of 1.

The test substance was applied by the installation of 0.057 g of the powdered test substance (with any chunks broken down with use of a spatula) into the everted lower lid of the right eye and then both lids were gently held together for 1 second. The left eye was used as an untreated control.

Readings of ocular irritation were performed at 1#, 24, 48, 72, 96 hours after application and again at 7, 14 and 21 days. Findings in the eyes were graded and scored according to the method of Draize.

# scores at this evaluation were not used in the criteria for classification according to Regulation (EC) 1272/2008.

 

According to Regulation (EC) 1272/2008 Eye Irritation Category 2 is based on:

Substances that produce in at least in 2 of 3 tested animals, a positive response of:

(a) corneal opacity ≥ 1 and/or

(b) iritis ≥ 1, and/or

(c) conjunctival redness ≥ 2 and/or

(d) 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

 

Cornea - scores were 0 at all time points.

Iris: A single score of 1 was recorded for animal #3 at 1 hour post installation. All other scores were 0.

Conjunctivae: When the mean grading of 24, 48 and 72 hours were calculated all mean scores for redness and chemosis were less than 2 and therefore the criteria for Irritation Category 2 were not met. When reversibility was taken into consideration the findings in animals #1 and #2 were fully reversible. For Animal #3, reversibility was demonstrated with a score of 0 on 2 consecutive occasions (96 hours and 7 days). Following this a score of 1 was reported on day 14 and 21; this was, therefore considered to be spurious. It may therefore be concluded that the findings were reversible.

From the findings in this study and according to the criteria described in Regulation (EC) 1272/2008, the criteria for classification were not met.

 

In an in vitro test, the test item and a 5% aqueous dilution of the test item were evaluated for eye irritation potential in the Isolated Chicken Eye (ICE) test according to OECD guideline 438 and given a reliability score of 1.

The test also included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 μL (liquids) or 30 mg (solids) for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed. 

The test item caused corneal effects consisting of very slight corneal swelling (mean of 4%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0). The opacity and fluorescein retention were observed as spots. In addition, yellow staining of cornea and sclera was observed which persisted until the end of the observation period. The 5% aqueous dilution of the test item only caused very slight corneal swelling (mean of 1%).

Since the test sample concerned a dye substance, the vitreous body of each eye was examined for possible staining during collection of the corneas in formalin and no staining was observed. Microscopic examination of the corneas treated with the test item revealed very slight erosion of the epithelium in two corneas. Microscopic examination of the corneas treated with a 5% aqueous dilution of the test item did not reveal any abnormalities.

 The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, pyknotic nuclei in the inner region of the stroma and necrosis of the endothelium.

Applying the classification criteria of the ICE, the following irritation classifications can be assigned to the test item:

-Category 2A:“Irritant/causes eye irritation” (UN-GHS classification);

-Category 2:“Irritating to eyes” (EU-CLP classification).

5% aqueous dilution of the test item:

-Not Classified (UN-GHS and EU-CLP classifications).

 

On the basis thatin vitroeye tests are not considered to be replacements for a well conducted guideline compliantin vivotest (ICCVAMhttp://europepmc.org/articles/pmc2849866) and in consideration of the potential for the ICEin vitrotest to give false positives at a rate of 11% for EU criteria (Test method evaluation report with details of ICCVAM recommendations and test method evaluation (2010), Table 5-2https://ntp.niehs.nih.gov/iccvam/docs/ocutox_docs/invitro-2010/tmer-vol1.pdf) it is concluded that the results from the OECD 405 in vivo eye irritation study should be used to predict the eye irritation potential of Vibracolor® Citrus Yellow. The results of this test demonstrate that the test item is not classified for eye irritation.

 

Justification for classification or non-classification

Vibracolor® Citrus Yellow is not classified for skin or eye irritation in well performed in vivo studies in the rabbit.