1,3-bis[3-(dimethylamino)propyl]urea

Endpoint summary

Description of key information

The test item was a mixture of the reaction products (1-(3-(Dimethylamino)propyl)urea (CAS 31506-43-1, EC 401-950-2, monoDMAPAU) )and 1,3-bis[3-(dimethylamino)propyl]urea (CAS  52338-87-1, EC 257-861-2, bisDMAPAU)

The test substance was evaluated with the Corrositex test method to determine its corrosive potential and to designate its Packing Group classification. The results of this study indicated that the sample was compatible with the Corrositex system. The pH of the test material placed it in Category 1. The results obtained from the evaluation of four replicate samples were reproducible, demonstrating that a mean time of 40.44 minutes was required to destroy the synthetic biobarriers.

In an in vivo skin irritation study irritation study, the test substance, after 3 minute exposure showed no erythema or edema. After 1 hour exposure there was no erythema or edema. After 4 hour exposure there was no erythema or edema.

Generally, results of in vivo studies have a higher relevance than those from in vitro studies.
Therefore the substance has not to be classified for skin irritation, based on the result of the in vivo study.

In an eye irritation study, corneal opacity, iritis and conjunctival irritation persisted through day 7. One instance of soiling of the anogenital area was noted during the observation period. monoDMAPAU is corrosive to a rabbit eye.

Based on the presence of approximately 15% of 1,3-bis[3-(dimethylamino)propyl]urea.(bisDMAPAU) in the test item, and according to the attached read-across justification this result is also deemed valid for 1,3-bis[3-(dimethylamino)propyl]urea (bisDMAPU).

bisDMAPAU is classified as causing serious Eye Damage Eye Category 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

26 June 2020

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: Sponsor; Batch No.: UA21111132
- Purity, including information on contaminants, isomers, etc.: not reported

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

Test system:

human skin model

Remarks:

EpiDerm™-Standard Model (EPI-200™): normal human epidermal keratinocytes (NHEK) which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstituted three-dimensional human epidermis model

Justification for test system used:

The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) whichmay be considered as non-irritant.

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of
normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDermTM (MatTek)
- Tissue batch number(s): 34191
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution: 1. MTT stock solution: 5 mg/mL MTT (VWR; Lot 20B2456894) in PBS (Gibco; Lot No.: 2306402); 2. MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Incubation time: 60 min
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability, Barrier function and Morphology: Tissue viability and the barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: The cells used to produce EpiDermTM tissue are screened for potential biological contaminants. Tests for each potential biological contaminant listed below were performed according to the test method give. Results of “Non detected” indicated that testing for the potential biological contaminant was not observed as determined by the stated test method.
NUMBER OF REPLICATE TISSUES: three
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates: three
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Mean Tissue Viability (% negative control) is <= 50 %-> Irritant: UN GHS -> Category 2
- Mean Tissue Viability (% negative control) is > 50% -> Non- Irritant -> UN GHS “No Category”

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 μL

Number of replicates:

Three tissues per dose group

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean Relative Tissue Viability [%]

Value:

3.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

Mean Relative Tissue Viability [%]: 100

Positive controls validity:

valid

Remarks:

Mean Relative Tissue Viability [%]: 3.9 %

Other effects / acceptance of results:

Pre-Experiments: The mixture of 30 μL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.
The mixture of 30 μL of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equaled 0%.
The test item showed non-specific reduction of MTT but no relevant colouring potential after mixturewith aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false negative results were necessary

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm^TM (MatTek) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study PU-2021-958 was applied topically to the EpiDerm^TM tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.
Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.
The test item showed non-specific MTT-reducing potential. The test item did not show water-colouring potential. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.
Since neither killed tissue controls nor controls with viable tissues were performed, no additional controls for correction of possible false-negative results were necessary.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was ≤ 50%. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The test item was a mixture of the reaction products (1-(3-(Dimethylamino)propyl)urea (CAS 31506-43-1, EC 401-950-2, monoDMAPAU) )and 1,3-bis[3-(dimethylamino)propyl]urea (CAS 52338-87-1, EC 257-861-2, bisDMAPAU), and urea in diproylene glycol.

Both source (1-(3-(Dimethylamino)propyl)urea (CAS 31506-43-1, EC 401-950-2) )and target 1,3-bis[3-(dimethylamino)propyl]urea (CAS 52338-87-1, EC 257-861-2) substances are based on the reaction product of dimethylaminopropylamine (DMAPA) and urea.
The main product of this reaction is the singly-substituted urea product, (3-(dimethylamino)propyl) urea (or mono-DMAPAU)
However, an unavoidable side-reaction is the formation of the doubly-substituted product, 1,3-bis[3-(dimethylamino)propyl]urea, which is formed by the disproportionation of mono-DMAPAU.
The test item will contain approximately 10% of 1,3-bis[3-(dimethylamino)propyl]urea.
Read-across is also claimed based on structural similarity and properties of both reaction products. Justification attached.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

This study was designed to comply with the standards set forth in OECD Guidelines For Testing Chemicals, Number 405, adopted February 24, 1987.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Source : Air Products and Chemicals, Inc.
Date Received : 12/17/99
Label Identity : NE 1060
Storage : Room temperature and humidity.
Description : Clear liquid
Sample Preparation : The test article was used as received.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

New Zealand White rabbits were received from Ace Animals, Boyertown, PA on 12/21/99 and quarantined for at least five days. Only animals in apparent good health were made available for study assignment. Prior to being selected for this study, both eyes of each animal were examined according to the Draize technique for any evidence of irritation or abnormalities of the cornea, iris and/or conjunctiva. A Mini-MagliteB flashlight equipped with a high intensity bulb was used to aid in the examination. Three rabbits (males), free from evidence of ocular irritation or abnormalities, were assigned to this study.

The animals were born the weeks of 10/10 through 10/24/99. The pretest body weight was 2.1 kg. The animals were identified by cage notation and a uniquely numbered metal eartag. The animals were housed llcage in suspended cages. Bedding was placed beneath the cages and changed at least three timeslweek. Fresh Purina Rabbit Chow (Diet #5321) was provided daily. Water was available ad libitum. The animal room, reserved exclusively for rabbits on acute tests, was temperature controlled, had a 12 hour lightldark cycle and was kept clean and vermin free.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

The test article (0.1 ml) was placed by syringe into the conjunctival sac which was formed by gently pulling the lower eyelid away from the eye.

Duration of treatment / exposure:

After instillation, the lids were held together for approximately one second to insure adequate distribution of the test article.

Observation period (in vivo):

Using a Mini-Maglit- flashlight equipped with a high intensity bulb, the treated eye of each rabbit was examined for irritation of the cornea, iris and conjunctiva at 1,24,48, and 72 hours post dose and on day 7. Ocular reactions were graded according to the numerical Draize technique. Additional signs were described.

Duration of post- treatment incubation (in vitro):

The eyes were examined and scored by the Draize technique at 1,24,48 and 72 hours post dose and on day 7

Number of animals or in vitro replicates:

Three rabbits (males), free from evidence of ocular irritation or abnormalities, were assigned to this study.

Details on study design:

Test Animals
New Zealand White rabbits were received from Ace Animals, Boyertown, PA on 12/21/99 and
quarantined for at least five days. Only animals in apparent good health were made available for study assignment. Prior to being selected for this study, both eyes of each animal were examined according to the Draize technique for any evidence of irritation or abnormalities of the cornea, iris and/or conjunctiva. A Mini-MagliteB flashlight equipped with a high intensity bulb was used to aid in the examination. Three rabbits (males), free from evidence of ocular irritation or abnormalities, were assigned to this study.
The animals were born the weeks of 10/10 through 10/24/99. The pretest body weight was 2.1 kg. The animals were identified by cage notation and a uniquely numbered metal eartag. The animals were housed llcage in suspended cages. Bedding was placed beneath the cages and changed at least three timeslweek. Fresh Purina Rabbit Chow (Diet #5321) was provided daily. Water was available ad libitum. The animal room, reserved exclusively for rabbits on acute tests, was temperature controlled, had a 12 hour lightldark cycle and was kept clean and vermin free.

Dosing
One eye of each rabbit was dosed. The contralateral eye sewed as a control. The test article (0.1 ml) was placed by syringe into the conjunctival sac which was formed by gently pulling the lower eyelid away from the eye. After instillation, the lids were held together for approximately one second to insure adequate distribution of the test article.

Type and Frequency of Observations
Using a Mini-Maglit- flashlight equipped with a high intensity bulb, the treated eye of each rabbit was examined for irritation of the cornea, iris and conjunctiva at 1,24,48, and 72 hours post dose and on day 7. Ocular reactions were graded according to the numerical Draize technique. Additional signs were described.
Body weights were recorded pretest.
The general health of the animals was monitored at each observation time.

Analysis of Data
The primary eye irritation score for each rabbit was calculated from the weighted Draize scale.
Eve irritation is the production of reversible changes in the eye following application of the test article to the anterior surface of the eye.
Eve corrosion is the production of irreversible tissue damage to the eye following application of the test article to the anterior surface of the eye.

Retention of Data
The raw data is filed at MB Research by project number. The final report is filed at MB Research by sponsor name and MB project number.
The test article will be returned to the sponsor following submission of the report.
Amendment to the Protocol
There were no amendments to the protocol

Irritation parameter:

cornea opacity score

Basis:

animal: #1,#2, #3

Time point:

7 d

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal: #1,#2, #3

Time point:

7 d

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: #1,#2, #3

Time point:

7 d

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritant / corrosive response data:

Corneal opacity, iritis and conjunctival irritation persisted through day 7.

Other effects:

Systemic Observations: One instance of soiling of the anogenital area was noted during the observation period.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Corneal opacity, iritis and conjunctival irritation persisted through day 7. One instance of soiling of the anogenital area was noted during the observation period.
NE 1060 is corrosive to the rabbit eye.
Based on the presence of approximately 10% of 1,3-bis[3-(dimethylamino)propyl]urea.(bisDMAPAU) in the test item, and according to the attached read-across justification this result is also deemed valid for 1,3-bis[3-(dimethylamino)propyl]urea (bisDMAPU).
bisDMAPAU is classified for serious eye damage - Eye cat 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)