1,3-bis(hydroxymethyl)urea

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2011 to 06 September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(hydroxymethyl)urea

EC Number:

205-444-0

EC Name:

1,3-bis(hydroxymethyl)urea

Cas Number:

140-95-4

Molecular formula:

C3H8N2O3

IUPAC Name:

1,3-bis(hydroxymethyl)urea

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, Kent CT9 4LT, United Kingdom
- Females nulliparous and non-pregnant: yes
- Age at study initiation: pre-test: 8 - 9 weeks, main study: 8 - 9 weeks
- Weight at study initiation: 16.1 - 20.1 g
- Housing: group, Makrolon Type II (pre-test)/ III (main study), with wire mesh top, granulated soft wood bedding
- Diet: ad libitum, pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 -100
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

5, 10, 25 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was a 25 % (w/w) suspension in dimethylsulfoxide as vehicle. At a concentration of 10 % and below, the test item could be dissolved in the vehicle. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonicating, warming to 37 °C).
- Irritation:
Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 10 % and 25 % (w/w) once daily each on three consecutive days.
- Systemic toxicity:
Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily.
- Ear thickness measurements:
Prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany)
- Erythema scores:
Ear irritation was considered to be excessive if reddening of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25 % was recorded on day 3 or day 6. The measured ear weight from biopsy punches was also considered in this evaluation.

On day 4 and day 5, both animals treated with 10 % or 25 % of the test item showed an erythema of the ear skin (Score 1). On day 3 and 4, slight swelling of the ears was observed in both treated animals. Signs of systemic toxicity were not observed during the period of the pre-test. However, neither the ear weight nor the ear thickness showed an increase above the threshold value for excessive local skin irritation of 25 % mentioned by the OECD Guideline 429. Thus, the test item in the main study was assayed at 5, 10 and 25 % (w/w).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The animals were distributed into the test groups at random and identified by cage number.
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index and if the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item preparations were made freshly before each dosing occasion.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation) as well as for the ear weights, lymph node weights and lymph node cell counts.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way- Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003).

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.08

Test group / Remarks:

5 %

Results 2

Key result

Parameter:

SI

Value:

1.03

Test group / Remarks:

10 %

Results 3

Key result

Parameter:

SI

Value:

1.21

Test group / Remarks:

25 %

Results 4

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control (DMSO)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
For the 5 % test substance group the mean DPM count was 901.4 ± 132.4. For the 10 % test substance group the mean DPM count was 859.6 ± 287.3. For the 25 % test substance group the mean DPM count was 1007.6 ± 307.1.

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index was determined by the ratio of proliferation in test item treated groups compared to that in vehicle controls.

EC3 CALCULATION
The EC3 value could not be calculated since all S.I.´s were below 3.

CLINICAL OBSERVATIONS:
The animals did not show any signs of systemic toxicity during the course of the study. From day 2 to 5, an erythema of the ear skin was observed (Score 1; in the groups treated with 5 and 25 % on day 2, at 5 % on day 3, at 5 and 25 % on day 4 and at all concentrations on day 5). Furthermore, slightly scaly ear skin was observed in the groups treated with 5 and 25 % on day 2, 4, and 5 and only on day 5 in the group treated with 10 %.

BODY WEIGHTS
The body weight of the animals recorded prior to the first application and prior to treatment with 3HTdR was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Lymph Node Weights and Cell Counts:

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. No statistically significant or biologically relevant increase in lymph node weight or lymph node cell count was observed in any dose group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold.

Ear Weights:

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. None of the indices determined for the test item treated groups exceeded this threshold.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo skin sensitisation assay (LLNA) in mice according to OECD Guideline 429, the test substance showed no skin sensitisation potential.

Executive summary:

A study according to OECD guideline 429 was conducted to assess the test substance for its skin sensitising potential. For this purpose a local lymph node assay in young adult female mice (CBA/CaCrl) was performed using test item concentrations of 5, 10, or 25 % (w/w) in the vehicle dimethylsulfoxide. The highest concentration tested was the highest concentration that could be used whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 2 to 5, an erythema of the ear skin was observed (Score 1; in the groups treated with 5 and 25 % on day 2, at 5 % on day 3, at 5 and 25 % on day 4 and at all concentrations on day 5). Furthermore, slightly scaly ear skin was observed in the groups treated with 5 and 25 % on day 2, 4, and 5 and only on day 5 in the group treated with 10 %. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. None of the indices determined for the test item treated groups exceeded this threshold. Stimulation Indices (S.I.) of 1.08, 1.03, and 1.21 were determined with the test item at concentrations of 5, 10 and 25 % (w/w) in dimethylsulfoxide, respectively. No statistically significant or biologically relevant increase in DPM value, lymph node weight, or lymph node cell count was observed in any dose group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold, confirming the above mentioned results. The test item was thus not a skin sensitiser under the test conditions of this study.