Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2016 to 04 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.405, "Acute Eye Irritation / Corrosion", Paris, 2012.

Deviations:

yes

Remarks:

Deviations from the maximum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B5: "Acute Toxicity: Eye Irritation/Corrosion". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

yes

Remarks:

Deviations from the maximum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.2400, Acute Eye Irritation. Office of Prevention, Pesticides and Toxic Items (7101), EPA 712-C-98-195, August 1998.

Deviations:

yes

Remarks:

Deviations from the maximum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF)

Version / remarks:

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions.

Deviations:

yes

Remarks:

Deviations from the maximum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

EC Number:

500-011-5

EC Name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Cas Number:

9003-80-9

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

N-phenylaniline; formaldehyde; propan-2-one

Test material form:

solid: flakes

Details on test material:

6.1. Test item
6.1.1. Test item information
Identification Formaldehyde, oligomeric reaction products with acetone
and diphenylamine
Appearance Dark brown flakes
Batch IC5B04P006
Purity/Composition 100% Unknown or Variable compositions, Complex reaction products and Biological materials (UVCB)
Test item storage At room temperature
Stable under storage conditions until 26 February 2019 (expiry date)

Specific details on test material used for the study:

Test substance: 206534/AIdentification: Formaldehyde, oligomeric reaction products with acetone and diphenylamineAppearance: Dark brown flakesBatch: IC5B04P006Purity/Composition: 100% Unknown or Variable compositions, Complex reaction products and Biological materials (UVCB)Test substance storage: At room temperatureStable under storage conditions until: 26 February 2019 (expiry date)Chemical name (IUPAC), synonym or trade name: Formaldehyde, oligomeric reaction products with acetone and diphenylamine (BXA)CAS Number: 9003-80-9pH (1% in water, indicative range): 7.76 – 7.37 (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 Males 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 labelled 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Animals were treated by instillation of, on average, 70.8 mg (range 70.8 – 70.9 mg) of the test item (a volume of approximately 0.1 mL),

Duration of treatment / exposure:

Single exposure

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 animals

Details on study design:

Study Design 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 3 weeks later, after considering the degree of eye irritation observed in the first animal. Preemptive 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 anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.Treatment Animals were treated by instillation of, on average, 70.8 mg (range 70.8 – 70.9 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 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. Immediately after fluorescein examination on Day 2, 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. After the final observation, the first animal was sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The other two animals were removed from the study alive.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.Necropsy: No necropsy was performed according to study plan.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

8

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Results 3

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Time point:

24/48/72 h

Score:

0.47

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Results 4

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Irritation Instillation of approximately 70.8 mg of Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 48 hours in one animal and within 72 hours in the other two 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 on the outside of the eyelids of all animals between Days 1 and 4. 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)	Fluor area (%)^2	(0-2)	Redness (0-3)	Chemosis (0-4)	Discharge (0-3)
121^1	1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	0	0 0 0 0	1 1 1 0	1 0 0 0	1 0 0 0	c - - -
138	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 1 0 0	c c c c
139	1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	0	0 0 0 0	2 1 1 0	1 0 0 0	1 1 0 0	c c c -

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

Comments:

c Remnants of the test item on the outside of the eyelids

 

Mean Value Eye Irritation Scores

Animal	Mean 24, 48 and 72 hours
	Corneal opacity	Iris	Conjunctivae
			Redness	Chemosis
121 138 139	0.0 0.0 0.0	0.0 0.0 0.0	0.7 0.0 0.7	0.0 0.0 0.0

 

Animal Specifications

Animal	Sex	Age at start (weeks)	Body weights (grams)
			Prior to application	At termination
121 138 139	M M M	14 12 12	3250 2613 2954	3335 2771 3024

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results, Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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.

 

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.

 

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 3 weeks later, after considering the degree of eye irritation observed in the first animal.

 

Single samples of approximately 70.8 mg of Formaldehyde, oligomeric reaction products with acetone and diphenylamine (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 approximately 70.8 mg of Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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 48 hours in one animal and within 72 hours in the other two animals.

 

Based on these results, Formaldehyde, oligomeric reaction products with acetone and diphenylamine 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).