6-methyl-2-oxoperhydropyrimidin-4-ylurea

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-01-10 to 2012-03-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BAYERISCHES LANDESAMT FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, Munich, Germany

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/CaOlaHsD
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 8-9 weeks
- Housing: in groups of 5 in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (lot no. 110811) in an air conditioned room (full barrier)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1114), ad libitum
- Water: tap water, sulfur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethyl sulphoxide

Concentration:

12.5, 25.0, and 50.0% (w/v) in vehicle

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 50% test item in DMSO was the maximum technically applicable concentration.
- Irritation: No remarkable changes in the ear thickness at the application site was detected in any of the animals, Thus, the test item was concluded to be not irritating under the conditions of the preliminary test.
- Systemic toxicity: No signs of systemic toxicity were observed in any of the animals.
- Body weights: All animals gained body weight as expected.
Based on the outcome of the reliminary test, the animals were treated with the test item up to the maximum technically applicable concentration (50% (w/v) in DMSO) in the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per min per lymph node (DPM/node) and as the ration of 3H-methyl thymidine-incorporation into lymph node cells of test group animals relative to that recorded for control group animals (Stimulation Index). Before DPM/node values were determined, background values were substracted.
EC3 values, calculated concentrations which induce Stimulation Indices of 3, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the Stimulation Indices axis, one above (a,b) and one below (c,d) the Stimulation Index of 3. If all measured points are above or below the Stimulation Index of 3, no EC3 value can be stated.
A substance is regarded as a skin sensitiser in the LLNA, if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine-incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals.

TREATMENT PREPARATION AND ADMINISTRATION:
- Dose groups: 3 test groups (3 different concentrations) and one negative control group (vehicle)were tested.
- Test regime:
Topical application: Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over 3 consecutive days.
Administration of 3H-methyl thymidine: 5 days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.
Preparation of cell suspension: Approximately 5 h after the injection of 3H-methyl thymidine all mice were sacrificed. The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation throughout polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspensio was pelleted in a centrifuge. The supernatan was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approximately 1 mL 5% trichloroacetic acid (TCA) at approximately 4 °C for approximately 18 h for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of incorporated 3H-methyl thymidine: The 3H-methyl thymidine -incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: p-Phenylendiamine (CAS 106-50-3, Sigma, purity > 98%; lot no. 060M0186V6), 1% in DMSO

Statistics:

linear interpolation for the calculation of EC3 values

Positive control results:

The positive control substance tested in the recent reliability check induced a Stimulation Index of 13.0±4.2.

Parameter:

SI

Remarks on result:

other: None of the 3 tested test material concentrations reached the Stimulation Index (SI) of 3. The following SI were calculated: - Negative control group: 1.0 - Test group (12.5%): 0.9±0.1 - Test group (25.0%): 0.8±0.2 - Test group (50.0%): 0.9±0.2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The following disintegrations per minute (DPM) were measured: - Negative control group: 3946.0, 3320.0, 3280.0, 3179.0, and 2743.0 (mean±SD: 3293.6±385.4) - Test group (12.5%): 2993.0, 2392.0, 2908.0, 3082.0, and 3448.0 (mean±SD: 2964.6±340.4) - Test group (25.0%): 2268.0, 3567.0, 1835.0, 3367.0, and 1924.0 (mean±SD: 2592.2±731.5) - Test group (50.0%): 2370.0, 3270.0, 4181.0, 2650.0, and 2836.0 (mean±SD: 3061.4±631.7)

Test results of the main study

- All animals survived throughout the test period without showing any clinical signs. There were test item residues at the application site at concentration of 50% after the application.

- All animals showed the expected body weight development.

- The means of the lymph node weights per group showed no significant difference compared to the negative control group.

Tab. 1: Radioactive Determination of the Test Substance Groups

Treatment	Concentration (%)	DPM/node (mean±SD)	SI	mean lymph node weight (g)
Negative control	-	1638.2±192.7	1.0	3.5
Test item in DMSO	12.5	1473.7±170.2	0.9±0.1	3.7
	25.0	1287.5±365.8	0.8±0.2	3.2
	50.0	1522.1±315.8	0.9±0.2	3.5

DPM/node= disintegrations per minute per lymph node

SI=Stimulation Index

Reliability Check:

The raw data of the reliability check are kept in the BSL archives (BSL Project ID 114612 K). The following results were obtained:

Tab. 2: Radioactive Determination of the Positive-Control Group of the Recent study

Treatment	DPM/node (mean±SD)	SI
Negative control	894.6±326.1	1
Phenylenediamine 1% in DMSO	11634.4±3771.3	13.0±4.2

DPM/node= disintegrations per minute per lymph node

SI=Stimulation Index

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the available key study (BSL, 2012d) the test item was investigated for skin sensitisation in the murine Local Lymph Node Assay, conducted according to the OECD test guideline 429, and in compliance with GLP. 25 µL of 12.5, 25.0, and 50.0% (w/v) test material solutions in DMSO were topically applied to the entire dorsal surface of each ear of 5 female CBA/CaOlaHsD mice per dose group. Concurrent negative control animals received the vehicle (DMSO) only. Topical applications were performed once daily over 3 consecutive days. 5 days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine (80 µCi/mL). Approximately 5 h after the injection of 3H-methyl thymidine all mice were sacrificed. The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared. The 3H-methyl thymidine -incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured. Determination of radioactivity was performed individually for each animal. All animals survived throughout the test period without showing any clinical signs. Furthermore, all animals showed the expected body weight development. The means of the lymph node weights per group showed no significant difference compared to the negative control group. None of the 3 applied test material concentrations reached the Stimulation Index (SI) of 3. For the test group groups SI of 0.9±0.1, 0.8±0.2, and 0.9±0.2 were calculated for 12.5, 25.0, and 50.0% test material concentrations, respectively. The positive control substance (P-Phenylendiamine) tested in the recent reliability check induced a Stimulation Index of 13.0±4.2, indicating that the test system used for this study is sensitive for the induction of skin sensitisation and that the negative result obtained in the present study is reliable.

Migrated from Short description of key information:
LLNA (OECD 429): not sensitising

Justification for selection of skin sensitisation endpoint:
The key study was selected for assessment.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No data available.

Migrated from Short description of key information:
No data available.

Justification for classification or non-classification

The available data are reliable and suitable for classification.Based on this data, classification for skin sensitisation according to Regulation (EC) 1272/2008 or Directive 67/548/EEC is not warranted.