2-(Hydroxyacetyl)-N-methylhydrazine-1-carboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th June 2017 to 25th July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: “The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds”, NIH No. 99-4494, 1999

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-N-[(methylcarbamoyl)amino]acetamide

EC Number:

815-593-9

Cas Number:

1613307-26-8

Molecular formula:

C4H10N3O3

IUPAC Name:

2-hydroxy-N-[(methylcarbamoyl)amino]acetamide

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J mice were received from Jackson Laboratories (Bar Harbor, ME) on 28 Jun 2017. Following an acclimation period of at least five days, fifteen nulliparous, non-pregnant female mice were assigned to the test groups without conscious bias.
The animals were born on 02 May 2017 (±3 days). The Day 1 body weight range for the study animals was 18.9 - 22.4 grams. The weight variation of the animals at study start did not exceed ±20% of the mean body weight. The animals were observed prior to the study start to ensure that no skin lesions were present on the ears.
The animals were identified by cage notation and indelible tail marks. During the acclimation period the animals were housed two per cage in suspended wire-bottom cages; during the study the animals were housed one per cage. Bedding was placed beneath the cages and changed at least three times per week. Fresh PMI Rodent Chow (Diet No. 5001) and water were available ad libitum. The animal room, reserved exclusively for mice on acute tests, was temperature- controlled, had a 12-hour light/dark cycle, and was kept clean and vermin free. Temperature and humidity were continuously monitored using automatic recording devices.

Study design: in vivo (LLNA)

Vehicle:

other: Dimethyl Sulfoxide (DMSO)

Concentration:

25% w/v

No. of animals per dose:

Five

Details on study design:

Groups of five CBA/J mice were treated by a topical application of a single test article concentration (25% w/v), vehicle control (DMSO) or positive control (25% HCA) to the dorsum of each ear once daily for three consecutive days. The test substance was spread over the entire dorsal surface of the ear using a micropipette to deliver 25 μl/ear.
All animals in the study were observed once daily throughout the study for clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality.
Body weights were recorded immediately prior to dosing on Day 1 and prior to euthanasia on Day 6. Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application (approximately 48 hours after the first test article application), and on Day 6 before euthanasia (approximately 120 hours after the first dose). Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25% or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.
Lymph Node Isolation and Processing
On Day 6, approximately 120 hours following the initial dose, and approximately five hours prior to euthanasia, the mice were injected with BrdU in Dulbecco's phosphate-buffered saline (DPBS) at a dose of 200 μl per mouse (15 mg/ml). The BrdU was administered by intraperitoneal injection. This thymidine analog becomes incorporated into the DNA of proliferating cells, including proliferating nodal lymphocytes. Each mouse was euthanized using CO2 asphyxiation, and the jugular vein was opened for complete exsanguination. Gross observations of the auricular lymph nodes were made, and the lymph nodes were collected. The auricular lymph nodes were combined for each animal and single-cell suspensions were generated in RPMI-10 medium and fixed with 85% ethanol. The cell suspensions were used to determine BrdU incorporation into the lymphocyte DNA (percentage of proliferating BrdU+ Lymph Node Cells [%BrdU+ LNC]) and the total number of cells in the nodes, for each individual animal.
Flow Cytometry
Flow cytometric analyses were conducted using a FACScan flow cytometer equipped with an Omnichrome argon laser emitting at 488 nm with 15 mW of power. For DNA and/or BrdU determinations, clumps of nuclei were excluded from analysis using gates set on integrated red fluorescence signals. The histograms generated in these experiments were analyzed using Cell Quest Software.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical evaluation of the calculated SI values was made by ANOVA followed by the Student's t-test, using GraphPad InStat™, version 3.06, 32 bit for Windows. However, determination of test article dermal sensitization was based on the criterion that a test article that yield an SI value of 3 or more is characterized as a potential sensitizing material.

Results and discussion

Positive control results:

SI = 4.5

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

25% (w/v)

Remarks on result:

not determinable

Cellular proliferation data / Observations:

The total number of proliferating lymphocytes was calculated by multiplying the total cell number by the percentage of the cells that have incorporated BrdU.
25% (w/v) L-005313063-000D009
Mean Total No. Cell in Node - 1776283
Mean %BrdU+ LNC - 0.85
Lymphocyte Proliferation (No. BrdU+) - 15047

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Topical application of test article L-005313063-000D009 at 25% (w/v) in the DMSO vehicle
resulted in an SI value less than 3 (SI < 3). Therefore, this test article is not a potential dermal sensitizer
in the Screening Local Lymph Node Assay.

Executive summary:

The test article was tested for solubility at 25% (w/v) in acetone:olive oil (4:1, AOO), dimethylacetamide:acetone:ethanol (4:3:3, DaAE), N,N-dimethylformamide, and dimethyl sulfoxide (DMSO). The test article was soluble only in DMSO; therefore, the Study Director chose DMSO as the vehicle for the study. One group of five healthy female CBA/J mice was treated with a 25% (w/v) concentration of the test article by topical application to the dorsum of each ear, once daily for three consecutive days. A vehicle control group of five mice was treated with DMSO, and another group of five mice was treated with the positive control, alpha-hexylcinnamaldehyde (85%) in DMSO (25% HCA), in the exact same manner.
The mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2’-deoxy-uridine (BrdU) approximately five hours prior to euthanasia. After euthanasia, the auricular lymph nodes were isolated, single-cell suspensions of lymph node cells (LNC) were generated, and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation (%BrdU+ LNC) and the total number of LNC. The amount of proliferating (BrdU+) LNC was determined as a measure of the proliferative response of the local lymph node. The stimulation index (SI) was calculated by dividing the proliferative response (number of BrdU+ LNC) of each test article-treated animal by the mean proliferative response of the vehicle control group. The mean SI and standard deviation (S.D.) was calculated for each group from the individual animal data. A test article treatment that yielded an SI value of 3 or more was characterized as sensitizing.

All animals survived the in-life phase of the study and were observed to be normal. Body weight changes were normal. Ear thickness measurements and individual animal observations indicated that the test article treatment did not result in dermal irritation.
The SI of the positive control, 25% HCA, was 4.5. The SI value for the test article at 25% (w/v) was 1.3.

Topical application of test article L-005313063-000D009 at 25% (w/v) in the DMSO vehicle resulted in an SI value less than 3 (SI < 3). Therefore, this test article is not a potential dermal sensitizer in the Screening Local Lymph Node Assay.