Registration Dossier

Skin Irritation:

The sum of the average stimulus values after 24 h (erythema = 1.3, edema = 0) and 72 h (erythema = 0.7, edema = 0) obtained by dividing by the number of these time points (by 2) gave a primary irritation score of 1.0.

From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

Eye Irritation:

The primary eye irritation score for 4-nitro-m-phenylenediamine was determined to be 0.0 .

From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be not irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

data is from experimental reports

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Principles of method if other than guideline:

To assess the dermal irritation potential of 4-nitrobenzene-1,3-diamine in rabbits

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material : 4-Nitro-m-phenylenediamine
- Molecular formula : C6H7N3O2
- Molecular weight : 153.14 g/mol
- Smiles notation : c1(c(cc(N)cc1)N)[N+](=O)[O-]
- InChl (if other than submission substance): 1S/C6H7N3O2/c7-4-1-2-6(9(10)11)5(8)3-4/h1-3H,7-8H2
- Substance type: Organic
- Physical state: Solid

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hacking & Churchill ltd, Huntingdon, UK
- Age at study initiation: Not reported
- Weight at study initiation: average about 3 kg
- Housing: The animals were housed individually
- Diet (e.g. ad libitum): "muemmel Z-222" (Hoeing Kraftfutterwerk Lower Saxony, Verden / Aller), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21°C
- Humidity (%): 40-50%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light / dark cycle (artificial lighting. 7 - 19 h)

IN-LIFE DATES: From: To: Not reported

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes

Amount / concentration applied:

0.5 g of creamed with water test substance were applied.

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48 and 72 h after removal of the lobules and conclusion of the test after 10 test day.

Number of animals:

3 male animals

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- % coverage: full
- Type of wrap if used: Gauze patch

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 4 hours

SCORING SYSTEM: As per the method of Draize

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24 hrs

Score:

1.3

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 72 hrs

Score:

0.3

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 72 hrs

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

After one hour, a yellowish discoloration of the skin was found. Discoloration held until the 9th day of the experiment at (Animal No. 4). The animals were followed up until the 10th day of the experiment,

	Time after patch removal	Skin irritation Animal No.			Total	Average
	Time after patch removal	3	4	5	Total	Average
Erythema	1 hr	1	3	1	-	-
	24 hr	1	2	1	4	1.3
	48 hr	1	2	1	-	-
	72 hr	1	1	0	2	0.7
	Experimental end	0	0	0	-	-

Edema	1 hr	0	0	0	-	-
	24 hr	0	0	0	0	0
	48 hr	0	0	0	-	-
	72 hr	0	0	0	0	0
	Experimental end	0	0	0	-	-

The sum of the average stimulus values after 24 h (erythema = 1.3, edema = 0) and 72 h (erythema = 0.7, edema = 0) obtained by dividing by the number of these time points (by 2) gave a primary irritation score of 1.0

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The sum of the average stimulus values after 24 h (erythema = 1.3, edema = 0) and 72 h (erythema = 0.7, edema = 0) obtained by dividing by the number of these time points (by 2) gave a primary irritation score of 1.0.
From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

Executive summary:

The primary skin irritation of 4-nitro-m-phenylenediamine was assessed in a study conducted on rabbits as per OECD Guideline 404.

The flank skin of 3 male New Zealand White rabbits was depilated on both sides on an area of approximately 6 x 6 cm with an electric clipper with suction for 24 hours before starting the test. In the experiment only animals with intact skin were taken. 0.5 g of 4 -nitro-m-phenylenediamine was applied under occlusion to clipped intact skin of the flank. The untreated side served as a control.The survey findings was performed 1, 24, 48 and 72 h after removal of the lobules and conclusion of the test after 10 test day. The stimulus values after 24 and 72 h were averaged over the experimental animals and dividing the resultant sum of the averages by 2.

After one hour, a yellowish discoloration of the skin was found. Discoloration held until the 9th day of the experiment at (Animal No. 4). The animals were followed up until the 10th day of the experiment.

The sum of the average stimulus values after 24 h (erythema = 1.3, edema = 0) and 72 h (erythema = 0.7, edema = 0) obtained by dividing by the number of these time points (by 2) gave a primary irritation score of 1.0.

From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

Endpoint conclusion:

adverse effect observed (irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

data is from experimental reports

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

To assess the ocular irritation potential of 4-nitro-m-phenylenediamine in rabbits

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material : 4-Nitro-m-phenylenediamine
- Molecular formula : C6H7N3O2
- Molecular weight : 153.14 g/mol
- Smiles notation : c1(c(cc(N)cc1)N)[N+](=O)[O-]
- InChl (if other than submission substance): 1S/C6H7N3O2/c7-4-1-2-6(9(10)11)5(8)3-4/h1-3H,7-8H2
- Substance type: Organic
- Physical state: Solid

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Sex: Male
- Source: Hacking & Churchill ltd, Huntingdon, UK
- Age at study initiation: Not reported
- Weight at study initiation: average about 3 kg
- Housing: The animals were housed individually
- Diet (e.g. ad libitum): "muemmel Z-222" (Hoeing Kraftfutterwerk Lower Saxony, Verden / Aller), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21°C
- Humidity (%): 40-50%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light / dark cycle (artificial lighting. 7 - 19 h)

IN-LIFE DATES: From: To: Not reported

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.1 g of the solid test sample was applied by single injection in the conjunctival sac of an eyelid.

Duration of treatment / exposure:

24 hrs

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 24 hours
After application, the eyelids for about 1 sec were gently held together. The other eye remained untreated as a control. After 24 hours the treated eye was rinsed with physiological saline solution.

SCORING SYSTEM: As per the method of Draize
The sum of the weighted vote degrees in all animals at 24, 48 and 72 h (s, 4.1, a + b + c) was divided by the sum of the number of animals and observation time points (9) and yielded the-primary irritation score.

TOOL USED TO ASSESS SCORE: ophthalmoscope
The observations on the cornea after 24 hours was secured by application of 1 drop of 1% fluorescein solution in ophthalmic formulation and subsequent rinsing with saline solution under UV light.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

72 h

Score:

0

Max. score:

9

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Of the controls differing findings were not charged. The animals were followed up until the 7th day of the experiment.Primary irritation score: 0/9

Table 1:Irritation Scores

Reading	1 hr			24 hr			48 hr			72 hr
Animal No.	18	19	10	18	19	10	18	19	10	18	19	10
CORNEA
A. Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
B. Area affected	0	0	0	0	0	0	0	0	0	0	0	0
a=AxBx5	0	0	0	0	0	0	0	0	0	0	0	0

IRIS	0	0	0	0	0	0	0	0	0	0	0	0
b=Ax5	0	0	0	0	0	0	0	0	0	0	0	0

CONJUNCTIVA
A. Redness	0	0	0	0	0	0	0	0	0	0	0	0
B. Chemosis	0	0	0	0	0	0	0	0	0	0	0	0
C.	0	0	0	0	0	0	0	0	0	0	0	0
c=(A+B+C)x2	0	0	0	0	0	0	0	0	0	0	0	0

Total = a+b+c	0	0	0	0	0	0	0	0	0	0	0	0

Interpretation of results:

other: not irritating

Conclusions:

The primary eye irritation score for 4-nitro-m-phenylenediamine was determined to be 0.0 .
From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be not irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

Executive summary:

The primary eye irritation of 4-nitro-m-phenylenediamine was assessed in rabbits in a study conducted as per OECD Guideline 405.

0.1 g of the solid 4 -nitro-m-phenylenediamine was applied by single injection in the conjunctival sac of 3 male New Zealand White rabbits. The untreated eye served as a control. After 24 hours the treated eye was rinsed with physiological saline solution.The observations on the cornea after 24 hours were secured by application of 1 drop of 1% fluorescein solution in ophthalmic formulation and subsequent rinsing with saline solution under UV light. The animals were observed for signs of irritation at 1, 24, 48 and 72 hours. The animals were followed up until the 7th day of the experiment.

The reactions were scored as per Draize method.The sum of the weighted vote degrees in all animals at 24, 48 and 72 h (s, 4.1, a + b + c) was divided by the sum of the number of animals and observation time points (9) and yielded the-primary irritation score.

The primary eye irritation score of for 4-nitro-m-phenylenediamine was determined to be 0.0 . From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be not irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no study available

Skin Irritation:

The primary skin irritation of 4-nitro-m-phenylenediamine was assessed in rabbits in a study conducted at Bayer AG Institute for Toxicology (Sustainability Support Service (Europe) AB has Letter of Access). The study was performed as per OECD Guideline 404.

The flank skin of 3 male New Zealand White rabbits was depilated on both sides on an area of approximately 6 x 6 cm with an electric clipper with suction for 24 hours before starting the test. In the experiment only animals with intact skin were taken. 0.5 g of 4 -nitro-m-phenylenediamine was applied under occlusion to clipped intact skin of the flank. The untreated side served as a control. The survey findings was performed 1, 24, 48 and 72 h after removal of the lobules and conclusion of the test after 10 test day. The stimulus values after 24 and 72 h were averaged over the experimental animals and dividing the resultant sum of the averages by 2. After one hour, a yellowish discoloration of the skin was found. Discoloration held until the 9th day of the experiment at (Animal No. 4). The animals were followed up until the 10th day of the experiment.

The sum of the average stimulus values after 24 h (erythema = 1.3, edema = 0) and 72 h (erythema = 0.7, edema = 0) obtained by dividing by the number of these time points (by 2) gave a primary irritation score of 1.0.

From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

Eye Irritation:

In different studies, 4-nitro-m-phenylenediamine has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits along with predicted data for target chemical and its structurally similar read across substances, 2 nitro p –phenylene diamine [CAS: 5307-14-2] and 4-nitroaniline [CAS: 100-01-6].

The primary eye irritation of 4-nitro-m-phenylenediamine was assessed in rabbits in a study conducted at Bayer AG Institute for Toxicology (Sustainability Support Service (Europe) AB has Letter of Access). The study was performed as per OECD Guideline 405.

0.1 g of the solid 4 -nitro-m-phenylenediamine was applied by single injection in the conjunctival sac of 3 male New Zealand White rabbits. The untreated eye served as a control. After 24 hours the treated eye was rinsed with physiological saline solution. The observations on the cornea after 24 hours were secured by application of 1 drop of 1% fluorescein solution in ophthalmic formulation and subsequent rinsing with saline solution under UV light. The animals were observed for signs of irritation at 1, 24, 48 and 72 hours. The animals were followed up until the 7th day of the experiment.

The reactions were scored as per Draize method. The sum of the weighted vote degrees in all animals at 24, 48 and 72 h (s, 4.1, a + b + c) was divided by the sum of the number of animals and observation time points (9) and yielded the-primary irritation score.

The primary eye irritation score for 4-nitro-m-phenylenediamine was determined to be 0.0 .

From the above results, the classification of 4-nitro-m-phenylene diamine was considered to be not irritating according to the E.C Commission's proposal on criteria for effects and irritants as set out in Annex VI II B 6 Directive.

This result is supported by the experimental study summarized in Fd Cosmet. Toxicol. Vol. 15, pp. 607-610. 1977; for the structurally similar read across substance, 2 nitro p –phenylene diamine [CAS: 5307-14-2]..The procedure for the eye tests was based on that prescribed in the Code of Federal Regulations (Title 16, Sec. 1500.42). 2 nitro p phenylenediamine was prepared as a 2.5% (w/v) solution or suspension. (Suspension in 0.5% aqueous gum tragacanth, containing 0.05% Na2SO3). Eleven groups of three rabbits were used to test solutions, each of which was instilled into one eye of each of three rabbits. The reactions were scored and categorized as + or - according to whether extrapolation of the result would be expected to give a positive or negative test for eye irritation based on the definition in the Code of Federal Regulations, Test for Eye Irritants (Title 16, Sec. 1500.42).

None of the three animals treated with 2 nitro p phenylene diamine showed any response to treatment, and although occasional transient mild conjunctival inflammation was observed. The reactions did not persist for more than 24 hr.

Thus, 2 nitro p phenylene diamine was considered to be not irritating to rabbit eyes.

These results are further supported by the experimental study summarized in ACTor (Aggregated Computational Toxicology Resource) - ACToR EPA database, 2011; for the structurally similar read across substance, 4-nitroaniline [CAS: 100-01-6].4 -nitroaniline was instilled into rabbit eyes and observed for signs of irritation (dose and duration not specified).

No signs of irritation were observed. Hence, 4-nitroaniline was considered not irritating to rabbit eyes.

Based on the available data for the target as well it’s read across substances, 4-nitro-m-phenylene diamine can be considered not irritating to eyes.Comparing the above annotations with the criteria of CLP regulation, test chemical can be classified under the category “Not Classified”.

Available studies for 4-nitro-m-phenylenediamine indicate that it is not likely to cause any irritation to eyes, but causes moderate irritation to skin.

Hence, 4-nitro-m-phenylene diamine can be classified under the category “Not Classified” for eyes and “Category 2” for skin as per CLP.