Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Endpoint summary

Description of key information

Skin Irritation: Not irritating using the in vitro human skin model.
Skin Corrosion: Not corrosive using the in vitro human three dimensional epidermal model.

Skin Irritation/Corrosion - in vivo (OECD Guideline 404): Not irritating to the skin of New Zealand white rabbits.
Eye Irritation: Not irritating using the in vitro BCOP test.

Eye Irritation (OECD Guideline 405): Not irritating using the in vivo acute method in New Zealand white rabbits.

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:

11 July 2016 to 05 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 skin 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 (BCOP 508841; skin corrosion 508839; skin irritation 508839), ex-vivo and in vivo tests) to determine the need for in vivo skin testing. It was concluded that there is need to perform this in vivo skin irritation study in rabbit in order to establish the possible skin irritating properties of the test item.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.404: "Acute Dermal Irritation / Corrosion", Paris, 2015.

Deviations:

yes

Remarks:

see "Any other information"

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B4: "Acute Toxicity: Dermal Irritation/Corrosion". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

yes

Remarks:

see "Any other information"

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.2500, Acute Dermal Irritation. Office of Prevention, Pesticides and Toxic Items (7101), EPA 712-C-98-196, August 1998.

Deviations:

yes

Remarks:

see "Any other information"

GLP compliance:

yes

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 names (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 Bsoch)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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 Cedex, 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 skin to be treated was intact and 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.

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

other: watery ethanol (50% v/v)

Controls:

not required

Amount / concentration applied:

Each animal was treated by dermal application of 0.5 grams of the test item.

Duration of treatment / exposure:

Four hours

Observation period:

72 hours

Number of animals:

3 animalsThe 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 20 days later, after considering the degree of skin irritation observed in the first animal.

Details on study design:

Test Item Preparation The test item was ground to a powder using a pestle and mortar prior to weighing and since the test item did not mix with water, the test item was moistened with watery ethanol (50% v/v) (water: Elix, Millipore S.A.S., Molsheim, France; ethanol: Merck, Darmstadt, Germany), immediately before application, to ensure close contact with the animal's skin. No corrections were made for the purity/composition of the test item, since the guidelines require a fixed amount that has to be applied.Treatment Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimetres (10x15 cm). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations. Each animal was treated by dermal application of 0.5 grams of the test item. The test item was moistened with 0.6 mL of the vehicle and applied to the skin of one flank, using a metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage#. Four hours after the application, the dressing was removed and the skin cleaned of residual test item using tap water. 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 application) and on the day of the final observation. Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test item. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.Necropsy: No necropsy was performed according to study plan.Histopathology: No histopathology was performed.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No skin irritation was caused by 4 hours exposure to Formaldehyde, oligomeric reaction products with acetone and diphenylamine.There was no evidence of a corrosive effect on the skin.

Other effects:

Colouration / Remnants: Following exposure, light grey staining of the treated skin area was noted after removal of the test item. No further staining of the treated skin by the test item was observed and no test item remnants were seen. Clinical signs / Mortality: No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual Skin Irritation Scores

Following exposure, light grey staining of the treated skin area was noted after removal of the test item

Animal	123^1			143			144
Time after exposure	Erythema (0-4)	Oedema (0-4)	Comments	Erythema (0-4)	Oedema (0-4)	Comments	Erythema (0-4)	Oedema (0-4)	Comments
1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	- - - -	0 0 0 0	0 0 0 0	- - - -	0 0 0 0	0 0 0 0	- - - -

¹Sentinel

 

Mean Value Irritation Scores

Animal	Mean 24, 48 and 72 hrs
	Erythema	Oedema
123 143 144	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	After the final observation
123 143 144	M M M	14 12 12	3192 2812 2686	3337 2883 2894

 

Interpretation of results:

GHS criteria not met

Conclusions:

No skin irritation was caused by 4 hours exposure to Formaldehyde, oligomeric reaction products with acetone and diphenylamine.

Executive summary:

Primary skin irritation/corrosion study with Formaldehyde, oligomeric reaction products with acetone and diphenylamine in the rabbit (4-hour semi-occlusive application).

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

OECD No.404, "Acute Dermal Irritation/Corrosion" (2015)

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

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

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

 

Three rabbits were exposed to 0.5 grams of Formaldehyde, oligomeric reaction products with acetone and diphenylamine, moistened with 50% watery ethanol by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure.

 

No skin irritation was caused by 4 hours exposure to Formaldehyde, oligomeric reaction products with acetone and diphenylamine.

 

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 skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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.

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.

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.

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.

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

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)

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.

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.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

8

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

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

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.

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

 

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

An in vitro skin irritation test with formaldehyde, oligomeric reaction products with acetone and diphenylamine using a human skin model was conducted. Skin tissue was moistened with 5 μl of Milli-Q water and 10.3 to 13.7 mg of formaldehyde, oligomeric reaction products with acetone and diphenylamine was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with formaldehyde, oligomeric reaction products with acetone and diphenylamine compared to the negative control tissues was 70%. Since the mean relative tissue viability for formaldehyde, oligomeric reaction products with acetone and diphenylamine was above 50% after 15 ± 0.5 minutes treatment.

The positive control had a mean cell viability of 8% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

It is concluded that this test is valid and that Formaldehyde, oligomeric reaction products with acetone and diphenylamine is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

 

Skin Corrosion

An in vitro skin corrosion test was conducted with formaldehyde, oligomeric reaction products with acetone and diphenylamine using a human three dimensional epidermal model (EpiDerm (EPI-200). The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour. Skin tissue was moistened with 25 μl of Milli-Q water and approximately 25 mg of the test substance was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 89% and 96%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive.

Finally, it is concluded that this test is valid and that formaldehyde, oligomeric reaction products with acetone and diphenylamine is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.

 

Skin Irritation/Corrosion - In vivo

A primary skin irritation/corrosion study was conducted with formaldehyde, oligomeric reaction products with acetone and diphenylamine in the rabbit (4-hour semi-occlusive application). Three rabbits were exposed to 0.5 grams of formaldehyde, oligomeric reaction products with acetone and diphenylamine, moistened with 50% watery ethanol by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure.

No skin irritation occurred after exposure to Formaldehyde, oligomeric reaction products with acetone and diphenylamine for 4 hours.

 

Eye Irritation

An evaluation of the eye hazard potential of formaldehyde, oligomeric reaction products with acetone and diphenylamine using the Bovine Corneal Opacity and Permeability test (BCOP test). Formaldehyde, oligomeric reaction products with acetone and diphenylamine was tested through topical application for approximately 240 minutes. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (345.5 to 387.1 mg).

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The individual in vitro irritancy scores for the negative controls ranged from -1.1 to 0.9. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 154 and above the historical positive control current mean plus two standard deviations. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Formaldehyde, oligomeric reaction products with acetone and diphenylamine did not induce ocular irritation according to both endpoints, and the mean in vitro irritancy score was -0.7 after 240 minutes of treatment.

Since formaldehyde, oligomeric reaction products with acetone and diphenylamine induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

 

Eye Irritation - in vivo

An acute eye irritation/corrosion study was conducted with formaldehyde, oligomeric reaction products with acetone and diphenylamine in the rabbit. The study was conducted in accordance with OECD Guideline 405.

 

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 for eye irritation.

Justification for classification or non-classification

Skin Corrosion/Irritation

The results an in vivo skin irritation test showed that formaldehyde, oligomeric reaction products with acetone and diphenylamine is not corrosive or irritating to the skin.

Eye Irritation

The results an in vivo eye irritation test showed that formaldehyde, oligomeric reaction products with acetone and diphenylamine is not irritating to the eyes.