N,N'-(3,3'-dimethylbiphenyl-4,4'-ylene)di(acetoacetamide)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 December 2013 and 23 January 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(3,3'-dimethylbiphenyl-4,4'-ylene)di(acetoacetamide)

EC Number:

202-111-1

EC Name:

N,N'-(3,3'-dimethylbiphenyl-4,4'-ylene)di(acetoacetamide)

Cas Number:

91-96-3

Molecular formula:

C22H24N2O4

IUPAC Name:

N-{2-methyl-4-[3-methyl-4-(3-oxobutanamido)phenyl]phenyl}-3-oxobutanamide

Details on test material:

Identification: Naphtol AS-G trocken
Other Name: N,N’-(3,3’-Dimethylbiphenyl-4,4’-ylene)di(acetoacetamide)
CAS-Number: 91-96-3
EINECS / EC-No.: 202-111-1
Purity: 99.1 % (w/w)
Expiry Date: 03 June 2015

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animals and Animal Husbandry:
Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.42 to 3.44 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A volume of 0.1 mL of the test item, which was found to weigh approximately 71 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye.

Duration of treatment / exposure:

Single application only. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released.

Observation period (in vivo):

1, 24, 48 and 72 hours following treatment

Number of animals or in vitro replicates:

Three New Zealand White male rabbits.

Details on study design:

Test Item Formulation and Experimental Preparation:
For the purpose of the study the test item was used as supplied. The absorption of the test item was not determined.

Measurement of pH:
The pH of the test item was determined prior to commencement of the study. A 10% w/w aqueous preparation of the test item was found to be pH 7 immediately and after 10 minutes.

Procedure:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anaesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.

A volume of 0.1 mL of the test item, which was found to weigh approximately 71 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to a six point scale.

Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

After consideration of the ocular responses produced in the first treated animal, two additional animals were similarly treated.

Scoring system:
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation according to Draize, J.H, 1977.

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Any clinical signs of toxicity, if present, were also recorded.

Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Interpretation of Results:
Data was summarized in tabular form, showing for each animal the irritation scores for the designated observation time, a description of the degree and nature of irritation, the presence of serious lesions and non-ocular effects. The scores of each animal at the following reading times (24, 48 and 72 hours) were used in calculating the respective mean values for each type of lesion.

The results were interpreted according to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: (All animals)

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0

Max. score:

4

Remarks on result:

other: No corneal effects were noted during the study in any animal

Results 2

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

73792

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0

Max. score:

2

Remarks on result:

other: No iridial inflammation

Results 3

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

73808

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0

Max. score:

2

Remarks on result:

other: No iridial inflammation

Results 4

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

73865

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Remarks on result:

other: Iridial inflammation was one hour after treatment.

Results 5

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Remarks:

73792

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Results 6

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Remarks:

73808

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 7

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Remarks:

73865

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Results 8

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

73792

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Results 9

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

73808

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Results 10

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

73865

Time point:

other: Mean of scores at 24, 48, 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Ocular Reactions:
Individual and mean scores for eye irritation are given in Table 1.

No corneal effects were noted during the study.

Iridial inflammation was noted in one treated eye one hour after treatment.

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Minimal conjunctival irritation was noted in all treated eyes at the 24 Hour observation and in one treated eye at the 48 Hour observation.

Two treated eyes appeared normal at the 48 Hour observation and one treated eye appeared normal at the 72 Hour observation.

No corrosive effects were noted during the study. The test item did not induce significant or irreversible damage to the rabbit eye.

Other effects:

Clinical Observations:
No clinical signs of toxicity were noted during the study.

Body Weight
Individual body weights and body weight change are given in Table 2.

All animals showed expected gain in body weight during the study.

Any other information on results incl. tables

Table 1: Eye Irritation Scores

Individual Scores for Eye Irritation

Rabbit Number and Sex	IPR	Evaluation interval	Corneal Opacity	Area of CorneaI Opacity	Iris	Conjunctivae
						Redness	Chemosis	Discharge
73792 Male	0	1 Hour	0	0	0	2	2	1
73808 Male	0		0	0	0	2	1	2
73865 Male	0		0	0	1	2	1	2
73792 Male		24 Hour	0	0	0	1	1	0
73808 Male			0	0	0	1	1	1
73865 Male			0	0	0	1	1	1
73792 Male		48 Hour	0	0	0	0	0	0
73808 Male			0	0	0	1	0	0
73865 Male			0	0	0	0	0	0
73792 Male		72 Hour	0	0	0	0	0	0
73808 Male			0	0	0	0	0	0
73865 Male			0	0	0	0	0	0

IPR=Initial pain reaction

Eye Irritation Scores (Table 1 Continued)

Mean Values after 24, 48 and 72 Hours

 

Rabbit Number and Sex	Number of available data points	Corneal Opacity	Iris	Conjunctivae
				Redness	Chemosis
73792 Male	3	0.00	0.00	0.33	0.33
73808 Male	3	0.00	0.00	0.67	0.33
73865 Male	3	0.00	0.00	0.33	0.33

 

Table 2: Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
73792 Male	2.51	2.55	0.04
73808 Male	2.42	2.44	0.02
73865 Male	3.44	3.53	0.09

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

According to the findings in this study Naphtol AS-G trocken does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Executive summary:

SUMMARY

The primary eye irritation potential of Naphtol AS-G trockenwas investigated according to a method compatible with OECD test guideline No. 405, EC Method B5, US EPA OPPTS 870.2400 and JMAFF, 2000. The test item was applied by instillation of 0.1 mL into the right eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation. 

 

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iritis, redness and chemosis of the conjunctivae. The individual mean scores for corneal opacity and iritis were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.33, 0.67 and 0.33 for reddening and 0.33 for all three animals for chemosis.

 

The instillation of Naphtol AS-G trockeninto the eye resulted in iridial inflammation and conjunctival irritation. These effects were reversible and were no longer evident 72 hours after treatment (end of the observation period). No abnormal findings were observed in the cornea of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No clinical signs of toxicity were observed.

 

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

 

According to the findings in this studyNaphtolAS-G trocken does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.