3-methyl-1,1-diphenylurea

Endpoint summary

Description of key information

Three GLP studies are available. 

EU method B.46 (In vitro skin irritation: reconstructed human epidermis model test)

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

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

All studies are GLP and Klimish score 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.01.-23.01.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: normal human epidermal keratinocytes- Tissue batch number(s): The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Ashland, USA); Lot No. 21611, kit CTEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°CMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1mg/ml- Incubation time: 3h- Spectrophotometer: Libra S22- Wavelength: 570nm- Modifications to validated SOP: M/48/3 (VUOS-CETA, 2011)NUMBER OF REPLICATE TISSUES: 3 for every concetrationCONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- Fresh tissues - N. of replicates : 3- Method of calculation used: NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)-The cut-off values for the prediction of irritation are given below; these values are stated in EU Method B.46., part 2.2. INTERPRETATION OF RESULTS: The test substance is considered to be irritant to skin in accordance with UN GHS 1 category 2:(i) if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %.The test substance is considered to have no category:(ii) if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 25 mgNEGATIVE CONTROL- PBS (phosphate buffered saline) prepared 06/01/2015, exp. 06/07/2015- Amount(s) applied (volume or weight): 25µLPOSITIVE CONTROL5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 110414ZSB, exp. 04/11/2015MEDIUMEPI-100-NMM-SIT/Maintenance Medium, MatTek, Lot No. 011515MHD, exp. 29/01/2015

Duration of treatment / exposure:

3h

Number of replicates:

3

Duration of treatment / exposure:

1h

Details on study design:

TEST SYSTEMThe reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. TEST PROCEDURE1. DIRECT MTT REDUCTION-FUNCTIONAL CHECK IN TUBESSome test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional check for this possibility is performed as follows: the test substance is added to mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.2. MTT TESTAfter pre-incubations (1 and 18±3hours), tissues were topically exposed to the test chemicals for 1 hour. Three tissues were used per every concentration of the test substance (C2), for the positive (PC) and negative (NC) controls. Tissues were then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium. After 24±2 hours incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18±2 hours. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank. EVALUATION OF RESULTSFor further classification the relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %. The cut-off values for the prediction of irritation are given below; these values are stated in EU Method B.46., part 2.2.

Irritation / corrosion parameter:

other: other: OD570

Value:

2.22

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Value:

109.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

25 mg of the test substance was added to 1.0 ml of MTT medium. Solution was incubated for 1 hour (37±, 5±1 % CO₂, humidified). The test substance did not cause change of colour.

MTT TEST

25 mg ofthe test substancewasplaced directly atop the previously moistened tissue, so the test substance will cover its entiresurface. Length of exposition was 60±5 min. After that the test substance was removed, tissues were rinsed and post-incubated.

After post-incubations the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, theblue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.

OD₅₇₀measuring was performed after 2-hour extraction with shaking.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test substance was 109.6% of negative control average value, i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model. The test substance is considered to have no category in regard to skin irritation.

Executive summary:

Test substance, Akardit,was assayed for the in vitro skin irritation in human epidermal model EpiDerm^TM.The test was performed according to theMethod B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4, (1), (3).

After pre-incubation of tissues, 25 mg ofthe testwasplaced directly atop to the previously moistened tissue so it covered all tissue surfaces. Length of exposition was 60 minutes. Three tissues were used for every concentration and controls.

After removal of the test substance from tissues, tissues were post incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, average viability of tissues treated by the test substance was 109.6%, i.e.viability was >50 %.

The effect of the test substancewasnegative inEpiDerm^TMmodel (the tissue was not damaged).

According to the classification criteria given in chapter 3.9., the test substance is considered to have no category in regard to skin irritation.

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:

16.02.-24.02.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152- Weight at study initiation: 2,4-2,8 kg- Housing: Conventional animal room – individually in metallic cages- Diet: Pelleted standard diet for experimental animals ad libitum- Water: Drinking tap water ad libitum (quality corresponding to the Regulation No.: 252/2004 Czech Coll. of Law) - Acclimation period: yesENVIRONMENTAL CONDITIONS- Room temperature: 20± 3°C, permanently monitored - Relative humidity: 30 – 70%, permanently monitored- Light period: 12 hour light/12 hour dark Study Time ScheduleAnimal supply: 03. 02. 2015Experimental part of study: 16. 02. – 24. 02. 2015Evaluation of results and final report elaboration: 24. 02. – 03. 03. 2015

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied: 0,1 g- Test substance: it was used in delivery form-pH approximately 7

Duration of treatment / exposure:

24h

Observation period (in vivo):

1, 24, 48, and 72 hours

Number of animals or in vitro replicates:

1 female/initial test2 females/confirmatory test

Details on study design:

APPLICATION OF THE TEST SUBSTANCE- The test substance was placed in the conjunctival sac of one eye of animal after gently pulling the lower lid away from the ball. CLINICAL OBSERVATIONThe eyes were examined at 1, 24, 48 and 72 hours after application. After recording the observations at 72 hours, the eyes of rabbit were examined with the aid of fluorescein and the ophthalmoscopy. The grades of ocular reaction for single animal (observation of conjunctivae, cornea and iris) were recorded at each examination. In the end of observation period the animals were sacrificed by injection of veterinary preparation T61 (1 mL/kg).TOOL USED TO ASSESS SCORE: fluorescein and ophthalmoscopy

Irritation parameter:

cornea opacity score

Basis:

animal: 7,8.10

Time point:

other: 24,48,72h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal: 7,8,10

Time point:

other: 24,42,72h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal: 7,8,10

Time point:

other: 24,48,72h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal: 7,8,10

Time point:

other: 24,48,72h

Score:

0

Max. score:

0

Table No. 5 – Summary results of reaction of treated eye(grades)

Animal No.	Ocular lesions	Time interval of examination
		1h	24h	48h	72h
7	Cornea Iris Conjunctivae Chemosis	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0
8	Cornea Iris Conjunctivae Chemosis	0 0 1 0	0 0 0 0	0 0 0 0	0 0 0 0
10	Cornea Iris Conjunctivae Chemosis	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0

Interpretation of results:

GHS criteria not met

Conclusions:

No changes were observed on eye at 1 hour after application: only in animals No. 8: Conjunctivae - Some blood vessels were hyperaemic (injected). No negative changes of eyes of all rabbits were observed for the rest of the study. During the observation period (24, 48 and 72 hours after application) no irritating effects on the eye was observed in all animals. No clinical signs of systemic intoxication were detected.Akardit, is not irritating to eye of rabbit.

Executive summary:

The test substance, Akardit, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

 

The test was performed according to the Method B.5 Acute Toxicity: Eye Irritation/Corrosion.Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

 

The test was performed initially using one animal (No. 7). Because no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals (No. 8 and No. 10).

 

No changes were observed on eye at 1 hour after application: only in animal No. 8: Conjunctivae -Some blood vessels were hyperaemic (injected).

No pathological changes of eyes of all rabbits were observed for the rest of the study. During the observation period (24, 48 and 72 hours after application) no irritating effects on the eye was observed in all animals.

No clinical signs of systemic intoxication were detected.

 

Evaluation of results after single application demonstrated that the test substance, Akardit,is not irritating to the eye of rabbit.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irriration

Only In vitro test was performed. The effect of the test substance was negative in EpiDerm^TMmodel (the tissue was not damaged).

Acute toxicity study by dermal route does not indicate skin irritation. (LD50>200 mg/kg/bw)

Eye irritation

After evaluation of the Bovine Corneal Opacity and Permeability Test (In vitro test) the test substance was not identified as a corrosive or severe irritant.This result was confirmed by examination of eye after single application of substance to the conjunctival sac of rabbit (In vivo test). The test substance was not irritating to the eye of rabbit, no clinical signs of systemic intoxication were detected.

Justification for selection of skin irritation / corrosion endpoint:
Only one study are available. The test was performed according to the Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT This study is GLP. Klimish score 1.

Justification for selection of eye irritation endpoint:
The study EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion) was selected. This study is In vivo study and GLP. Klimish score 1.

Justification for classification or non-classification

Based on the available data, the substance Akardit is not classified to skin and eye irritation.