Urea, reaction products with diethylene glycol, formaldehyde and glyoxal

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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:

1-{[4,5-dihydroxy-3-(hydroxymethyl)-2-oxoimidazolidin-1-yl]methyl}-4,5-dihydroxy-3-(hydroxymethyl)imidazolidin-2-one; 2-(2-hydroxyethoxy)ethan-1-ol; 4,5-dihydroxy-1,3-bis({[2-(2-hydroxyethoxy)ethoxy]methyl})imidazolidin-2-one

EC Number:

939-056-4

Cas Number:

1469982-95-3

Molecular formula:

UVCB substance

IUPAC Name:

1-{[4,5-dihydroxy-3-(hydroxymethyl)-2-oxoimidazolidin-1-yl]methyl}-4,5-dihydroxy-3-(hydroxymethyl)imidazolidin-2-one; 2-(2-hydroxyethoxy)ethan-1-ol; 4,5-dihydroxy-1,3-bis({[2-(2-hydroxyethoxy)ethoxy]methyl})imidazolidin-2-one

Details on test material:

- Name of test material (as cited in study report): Fixapret PC, vor Magnesiumchlorid-Zugabe
- Physical state: liquid
- Analytical purity: Reaction product of urea, formaldehyde, glyoxal and diethylene glycol, content: 62.1 % in water
- Impurities (identity and concentrations): 37.9 % water (solvent)
- Lot/batch No.: 153
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor,
and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature; avoid temperatures < 5°C >30°C

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V.
- Age at study initiation: pretest: 9-10 weeks, main study: 8-9 weeks
- Weight at study initiation: 18,3 - 23,1 g
- Housing: 5 animals per cage
- Diet: ad libitum:
- Water: ad libitum:
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- Humidity (%): 44 – 68%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25, 50 and 100 %

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: up to 100 % (undiluted)
- Irritation: none

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

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, for
the ear weights, the lymph node weights and lymph node cell count, and for the DPM
values (group mean DPM ± standard deviation).
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).
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The periodic positve control gave the expected results.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle: 1 25 %: 0.57 50 %: 0.74 100 %: 0.89

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle: 757 DMP (mean) 25 %: 432.2 DMP (mean) 50 %: 562 DMP (mean) 100 %: 671.8 DMP (mean)

Any other information on results incl. tables

In this study the test item Fixapret PC, vor Magnesiumchlorid-Zugabe was assessed for its

skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item

solution at different concentrations was prepared in the vehicle N,N-dimethylformamide.

The local lymph node assay is recommended by international test guidelines (e.g. OECD)

as an animal test for predicting skin sensitization in humans and provides a rational basis

for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a

primary proliferation of lymphocytes in the lymph node draining the application site. The

ratio of proliferation in test item treated groups compared to that in vehicle controls is

termed the Stimulation Index (S.I.). Radioactive labelling is used to measure cell

proliferation.

For this purpose a local lymph node assay was performed using test item concentrations of

25, 50% (w/w), and 100% (undiluted test item).

The animals did not show any signs of systemic toxicity or local skin irritation during the

course of the study and no cases of mortality were observed. A statistically significant or

biologically relevant increase in ear weights was not observed in any treated group in

comparison to the vehicle control group. Furthermore, the cutoff-value for a positive

response regarding the ear weight index of 1.1 reported for BALB/c mice (see Ref. 9) was

not exceeded in any dose group.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item

concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared

with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test

item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 0.57, 0.74, and 0.89 were determined with the test

item at concentrations of 25, 50% (w/w) and 100% in N,N-dimethylformamide, respectively.

An EC3 value could not be calculated, since none of the tested concentrations induced a

S.I. greater than the threshold value of 3.

A statistically significant relevant increase in DPM value was observed in the low dose

group in comparison to the control group. However, this was not considered as biologically

relevant as the S.I. determined for this concentration did not exceed the threshold value of

3 mentioned by the OECD guideline 429. A statistically significant or biologically relevant

increase in lymph node weight and -cell count was not observed in any of the tested dose

groups. Furthermore, the the cutoff-value for a positive response regarding the lymph node

cell count index of 1.55 reported for BALB/c mice (see Ref. 8) was not exceeded in any

group.

The test item Fixapret PC, vor Magnesiumchlorid-Zugabe was thus not a skin sensitiser

under the test conditions of this study.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU