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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 May 2016 to 29 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
Deviations from the maximum daily mean relative humidity occurred.
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
Deviations from the maximum daily mean relative humidity occurred.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
yes
Remarks:
Deviations from the maximum daily mean relative humidity occurred.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-(2-methylpropylidene)bis[4,6-xylenol]
EC Number:
251-394-8
EC Name:
2,2'-(2-methylpropylidene)bis[4,6-xylenol]
Cas Number:
33145-10-7
Molecular formula:
C20H26O2
IUPAC Name:
2-[1-(2-hydroxy-3,5-dimethylphenyl)-2-methylpropyl]-4,6-dimethylphenol
Test material form:
solid: particulate/powder
Details on test material:
Test Item: 207366/A
Identification: Lowinox®22IB46
Appearance: White to cream coloured powder
Batch: WB44L0016
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 18 November 2018 (retest date)
Chemical name (IUPAC): 2,2’-(2-methylpropylidene)bis[4,6-xylenol]
CAS Number: 33145-10-7
pH (1% in water, indicative range): 8.0 – 7.2
Specific details on test material used for the study:
Chemical name (IUPAC), synonym or trade name: 2,2’-(2-methylpropylidene)bis[4,6-xylenol]
CAS Number: 33145-10-7
pH (1% in water, indicative range): 8.0 – 7.2 (determined by Charles River Den Bosch)

Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Species: Albino rabbit, New Zealand White, (SPF-Quality). Recognized by international guidelines as the recommended test system (e.g. EC, OECD)
Source: Charles River France, L’Arbresle, France
Number of animals: 3 Females.
Age and body weight: At start of dosing, the animals were between 12 and 24 weeks old and body weights were at least 1.5 kg.
Identification: Earmark.
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the eyes were free from any abnormality.
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod was between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
Acclimatization period was at least 5 days before start of treatment under laboratory conditions.
Diet: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.
Water: Free access to tap water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
Animals were treated by instillation of, on average, 25.9 mg (range 25.8 – 25.9 mg) of the test item (a volume of approximately 0.1 mL).
Duration of treatment / exposure:
Single dose, exposure time 1 hour.
Observation period (in vivo):
24 hour observation period
Number of animals or in vitro replicates:
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner one week later, after considering the degree of eye irritation observed in the first animal.
Details on study design:
Weight of Evidence: Analysis In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed, prior to the start of this in vivo eye irritation study in the rabbit. As recommended in the test guidelines, all available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity) and in vitro, ex vivo and in vivo tests) to determine the need for in vivo eye testing.
Based on the available information, it was concluded that there was the need to perform this in vivo eye irritation study in rabbit in order to establish the possible eye irritating properties of the test item.

Pre-emptive Pain Management: One hour prior to instillation of the test item, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia. Five minutes prior to instillation of the test item, two drops of the topical anaesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.
Treatment: Animals were treated by instillation of, on average, 25.9 mg (range 25.8 – 25.9 mg) of the test item (a volume of approximately 0.1 mL), in the conjunctival sac of one of the eyes 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 item. The other eye remained untreated and served as the reference control. Immediately after the 1 hour observation, the treated eye was rinsed with approximately 50 mL tepid tap water, using a velocity of flow which did not affect the eye, to remove any visible residual test item. For reference control the other eye was also rinsed. In order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection. 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 each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
Observations
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body Weight: Day of treatment (prior to instillation) and after the final observation.
Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to the following numerical scoring system. (see Any other information for details).
Necropsy: No necropsy was performed according to study plan.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 24 hours
Remarks:
see Irritant/corrosive data
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 24 hours
Remarks:
see Irritant/corrosive date
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Irritation: Instillation of approximately 26 mg of Lowinox® 22IB46 (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge.
The irritation had completely resolved within 24 hours in all animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.
Corrosion: There was no evidence of ocular corrosion.
Other effects:
Colouration / Remnants: Remnants of the test item were present in the eye on Day 1. Toxicity / Mortality No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Any other information on results incl. tables

Individual Eye Irritation Scores

Animal

Time after dosing

Cornea

Iris

Conjunctivae

Comments

Opacity

(0-4)

Area

(0-4)

Flour area (%)2

(0-2)

Redness

(0-3)

Chemosis

(0-4)

Discharge

(0-3)

821

1 hour

24 hours

48 hours

72 hours

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

1

0

0

0

1

0

0

0

b

-

-

-

93

1 hour

24 hours

48 hours

72 hours

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

1

0

0

0

1

0

0

0

b

-

-

-

94

1 hour

24 hours

48 hours

72 hours

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

1

0

0

0

1

0

0

0

b

-

-

-

1Sentinel,2Green staining after fluorescein treatment (percentage of total corneal area)

Comments:

b – Remnants of the test item in the eye.

 

Mean Value Eye Irritation Scores

Animal

Mean 24,48 and 72 hours

Corneal opacity

Iris

Conjunctivae

Redness

Chemosis

82

93

94

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

 

Animal Specifications

Animal

Sex

Age at start (weeks)

Body weights (grams)

Prior to application

At termination

82

93

94

F

F

F

22

23

23

3620

3671

4154

3752

3688

4295

 

Applicant's summary and conclusion

Interpretation of results:
other: No classified
Conclusions:
Based on the results, Lowinox® 22IB46 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).
Executive summary:

Acute eye irritation/corrosion study with Lowinox® 22IB46 in the rabbit.

 

The study was carried out based on the guidelines described in:

OECD No.405 (2012) "Acute Eye Irritation / Corrosion"

EC, No 440/2008, B5: "Acute Toxicity: Eye Irritation/Corrosion"

EPA, OPPTS 870.2400 (1998): "Acute Eye Irritation"

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

 

Single samples of approximately 26 mg of Lowinox® 22IB46 (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation.

 

Instillation of the test item resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 24 hours in all animals.

 

Remnants of the test item were present in the eye on Day 1.

 

Based on the results, Lowinox® 22IB46 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

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