Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Mar. 14, 2002 to Mar. 14, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline, well-documented study report with deviation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: Hen's egg test on the chorio-allantoic membrane (HET-CAM) assay - (INVITTOX Protocols 47& 96)
Deviations:
yes
Remarks:
Negative control was not included in the study
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methyl-p-phenylenediamine sulfate
EC Number:
210-431-8
EC Name:
2-methyl-p-phenylenediamine sulfate
Cas Number:
615-50-9
Molecular formula:
C7H10N2.H2O4S
IUPAC Name:
2-methyl-p-phenylenediamine sulphate
Constituent 2
Reference substance name:
2-methyl-p-phenylenediamine sulphate
EC Number:
228-871-4
EC Name:
2-methyl-p-phenylenediamine sulphate
Cas Number:
6369-59-1
IUPAC Name:
2-methylbenzene-1,4-diamine sulfate
Details on test material:
- Name of test material: p-toluylendiamin sulfate-Colipa A5-pTD - TSIN: SAT 010935- Substance type: Pure active substance- Physical state: Beige powder- Solubility: Slightly solubility in water, ethanol, DMSO, Paraffin (<1%)- Storage condition: Room temperature, dark

Test animals / tissue source

Species:
other: White Leghorn chicken
Strain:
other: Starcross 288

Test system

Vehicle:
water
Remarks:
distilled
Amount / concentration applied:
TEST MATERIAL- Amount applied : The following amount of test material was tested in Preliminary and main testsPreliminary study: Undiluted (99.9%), 25% suspension in water (reddish brown suspension) and aqueous solution (<1% beige)Main study: Undiluted (99.9%), The undiluted solid test substance was applied by the aid of a spatula, the diluted test substance was applied in a dose of 300 µL by the aid of a pipette which was sufficient to cover at least 25% of the membrane.VEHICLE: Vehicle, distilled water, was not applied to chorioallantoic membraneREFERENCE SUSBATNCE: Sodium magnesium laurymyristyl-6-ethoxy-sulfate (Texapon ASV 70)- Amount(s) applied: 300 µL- Concentration: 0.5, 1 and 5% - Lot/Batch No.: Batch # 90950015
Duration of treatment / exposure:
180 sec
Observation period (in vivo):
180 sec
Details on study design:
SOURCE OF EGGS: White leghorn chicken (source: Horst, D-51377 Leverkusen)MAINTENANCE OF EGGS BEFORE TEST TREATMENT: The Fresh, intact fertilized chicken eggs were maintained under following environmental conditions prior to the start of test: - Temperature (°C): 37.6 ±1 °C (incubator: Ehret, type KMB 4) - Humidity: Approx. 40-60%.NUMBER OF EGGS USED: Preliminary study: At least 2; Main study: At least 6TESTING PROCEDURES: The chorioallantoic membrane of fertilized hen’s eggs was used after an incubation time of 9 d under standard conditions. After the incubation period all eggs were checked with a candling lamp. Only eggs with a well developed embryonie vascular System were taken for the study. During candling the airspace outlined at the blunt end of the eggs was marked. These eggs were opened along the marked line with an electric drill. Then the white egg membrane was moistened with a few milliliters of physiological saline. If necessary the opened eggs were placed under a thermal lamp, in order to prevent cooling, until the test substance was applied. Prior the application, the egg membrane had to be removed from the chorioallantoic membrane.REMOVAL OF TEST SUBSTANCE- Washing (if done): Treatment was terminated by rinsing the chorioallantoic membrane with physiological saline.- Time after start of exposure: 180 secondsSYMPOTMS OBSERVED FOR THE EVALUATION OF IRRITATION EFFETC ON CHORIOALLANTOIC MEMBRANE (CAM): The following symptoms are the relevant irritation effects on the CAM:- Haemorrhage (bleeding out of capillaries)- Coagulation (Protein denaturation inside and/or outside the blood vessels)- Lysis (disintegration of blood vessels)END-POINT ASSESSMENT: If a non-transparent substance hinders continuous observation of the CAM, an end-point assessment was performed. In the end-point assessment the irritation categories of strength “slight”, “moderate” and “strong” were defined after an exposure period of five minutes in association with the active substance content of 0. 5, 1 and 5 % from “Texapon ASV”.SCORING SYSTEM: Sum of single value of either hemorrhage, vessel lysis or coagulation were calculated for each egg and scored as follows: 1. Slightly irritating: 0-5 (sum of single value) 2. Moderately irritating: 6-12 (sum of single value) 3. Irritating: 13-15 (sum of single value) 4. Severely Irritating: 16-18 (sum of single value)The irritation potential was determined in comparison to the internal reference substance Texapon ASV

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
coagulation
Run / experiment:
undiluted p-toluylendiamin sulfat applied by the aid of a spatula in a dose of 300 µL. It covers 25% of the chorioallantoic membrane of fertilised hen's egg. measurement after 180 seconds
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
control substance Texapon ASV 5% AS after 30 seconds
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Remarks:
vessel lysis
Run / experiment:
undiluted p-toluylendiamin sulfat applied by the aid of a spatula in a dose of 300 µL. It covers 25% of the chorioallantoic membrane of fertilised hen's egg. measurement after 180 seconds
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
control substance Texapon ASV 5% AS after 30 seconds
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Remarks:
haemorrhage
Run / experiment:
undiluted p-toluylendiamin sulfat applied by the aid of a spatula in a dose of 300 µL. It covers 25% of the chorioallantoic membrane of fertilised hen's egg. measurement after 180 seconds
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
control substance Texapon ASV 5% AS after 30 seconds
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Range of historical values if different from the ones specified in the test guideline:
OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Range of historical values if different from the ones specified in the test guideline:

Any other information on results incl. tables

Table 1: Scores forhemorrhage, vessel lysis, and coagulation after test substance exposure for 180 sec (study # 69835)

Egg number

Hemorrhage

Vessel lysis

Coagulation

1

-

-

-

2

-

-

-

3

-

-

-

4

-

-

-

5

-

-

-

6

-

-

-

Sum

-

-

-

Table 2: Scores forhemorrhage, vessel lysis, and coagulation after reference substance (5%) exposure for 30 sec (study # 69835)

Egg number

Hemorrhage

Vessel lysis

Coagulation

1

++

++

-

2

++

+

-

3

++

+

-

4

++

++

-

5

++

+

-

6

++

++

-

Sum

12

9

0

+: Slight

++: Moderate

+++: Strong

Applicant's summary and conclusion

Interpretation of results:
slightly irritating
Remarks:
Migrated informationCriteria used for interpretation of results: expert judgment
Conclusions:
Undiluted 2 -methyl-p-phenylenediamine sulphate was categorized as ‘slightly irritating’, compared to the reference substance, “Texapon ASV” (5%), using the HET-CAM (Hen’s Egg Test – Chorioallantoic Membrane) method.
Executive summary:

2 -methyl-p-phenylenediamine sulphate was tested for its acute eye irritation potential on the choriollantoic membrane (CAM) of fertilized chicken eggs.

Fresh, intact fertilized eggs of White Leghorn chicken were incubated for 9 d at temperature of 37.6±1°C and 40 -60% relative humidity. After a 9 d incubation period all eggs were checked with a candling lamp. Only eggs with a well developed embryonic vascular system were taken for the study. During candling the airspace outlined at the blunt end of the eggs was marked. The eggs were opened along the marked line with an electric drill. Then the white egg membrane was moistened with a few milliliters of physiological saline. If necessary the opened eggs were placed under a thermal lamp, in order to prevent cooling, until the test substance was applied. Prior to the test application, the egg membrane had to be removed from the CAM.

The test material was applied for 180 sec at the following concentration in the preliminary and main study:

Preliminary study (conducted with two eggs): Undiluted (99.9%), 25% suspension in water (reddish brown suspension) and aqueous solution (<1% beige)

Main study (conducted with six eggs): Undiluted (99.9%)

The undiluted solid test substance was applied by the aid of a spatula, the diluted test substance was applied in a dose of 300 µL by the aid of a pipette which was sufficient to cover at least 25% of the membrane. Sodium magnesium laurymyristyl-6-ethoxy-sulfate (Texapon ASV 70) (0.5, 1 and 5%) was used as reference substance.

The following symptoms were observed for the evaluation of irritation effects on the CAM:

     - Haemorrhage (bleeding out of capillaries)

     - Coagulation (Protein denaturation inside and/or outside the blood vessels)

     - Lysis (disintegration of blood vessels)

Treatment was terminated by washing of the CAM with physiological saline

The following classification scheme of irritating properties was defined for S-values:

End-Point Assessment

Exposure time:30 seconds/S[sum of single value]

Evaluation

0-5

Slightly irritating

6-12

Moderately irritating

13-15

Irritating (R 36)

16-18

Severely irritating (R 41)

 

In a pre-experiment neither the undiluted solid test substance induced any reaction in the end-point assessment after 30 and 180 sec. exposure time, nor did a 25% suspension or the aqueous solution (<1% in water). Hemorrhage, vessel lysis and coagulation scores were 0 for each egg in the main test.

In order to compare the results and to minimize effects due to biological variation of the egg batches, an irritation index of the reference substance "Texapon ASV" was calculated [5% active substance, pH 7]. This concentration was classified as moderately irritating to rabbit's eyes in the in vivo Draize test.

The test substance results in comparison to the reference substance is as follows:.

Substance

Active substance

Concentration

End-Point Method [S]

Evaluation

Test substance

99.9 %

99.9 %

0

Slightly irritating

Reference substance

68.5 %

5 % AS

12

Moderately irritating

Based on the results, undiluted 2 -methyl-p-phenylenediamine sulphate was categorized as ‘slightly irritating’, compared to the reference substance, “Texapon ASV” (5%), using the HET-CAM (Hen’s Egg Test – Chorioallantoic Membrane)method.