Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
end on 13-SEP-2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. However a corrosive effect was observed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
modified as IMDS test
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Uronium hydrogen sulphate
EC Number:
244-343-6
EC Name:
Uronium hydrogen sulphate
Cas Number:
21351-39-3
IUPAC Name:
amino(hydroxy)methaniminium hydrogen sulfate
Details on test material:
- Name of test material (as cited in study report): aduct urea-sulfuric
- Physical state: liquid
- Stability under test conditions: no data
- Storage condition of test material: dry, ventilated well, away from inflammable materials

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from the company Charles River Deutschland GmbH, D-97633 Sulzfeld
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 26 to 32 g
- Housing: transparent macrolone cages (type 150, floor area 810 cm2) with 6 animals in each cages
- Diet: a pelleted complete rodent diet "Altromin 1324", ad libitum
- Water: free access to bottles of drinking water of domestic quality, which was acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3°C
- Humidity: at least 30% and preferably not exceed 70%
- Air changes: 10 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: the experimental phase was carried out between July 20th, 2010 and July 23th, 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100% (v/v)
No. of animals per dose:
6 animals per dose
Details on study design:
RANGE FINDING TESTS: no pre-test was performed. The dose levels were chosen based on existing acute toxicity and dermal irritation (with conclusion that the test substance was not acutely toxic and not irritant to the skin in the rabbit).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA / IMDS
- Criteria used to consider a positive response:
a differentiation index (DI) is calculated from the different parameters of investigation which describes the relation between the activation of the local skin-draining lymph nodes and the skin inflammation at the site of topical treatment as well as it contributes to distinguish between inflammatory (non-specific) and allergic (specific) reaction:
- 0 < DI < 1 : inflammation
- DI > 1 : allergic reaction

DI = (% max. LN cell count index increase) / (% max. ear thickness increase)

TREATMENT PREPARATION AND ADMINISTRATION:
Twenty-four albino mice of the strain Crl:NMRI in groups consisting of six animals each were treated with three concentrations of the test item (100%, 50% and 25% v/v) or only with the vehicle acetone/olive oil (4/1 v/v) on three consecutive days. An amount of 25µL of the test substances was applied on the dorsal side of each ear.
Positive control substance(s):
not specified
Statistics:
data not available

Results and discussion

Positive control results:
The last positive test results was registered in August 2009 with differentiation index (DI) of 1.63 and 1.64

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see table

Any other information on results incl. tables

In contrast to the information known on the substance (i.e. no irritation by skin contact in the rabbit, no irritation observed in the acute oral and dermal test) a severe irritant effect of the test item Aduct urea-sulfuric was revealed.

On day 3 of the study, only a few animals of the test groups with concentrations of 50% and 25% showed initially well defined irritation in the form of necrotic changes of the ear skin. But on the end of the study (day 4), a damage of the epidermis was discernible in almost all animals (14 out of 18 mice) of the three test groups.

 

This observation of a severe irritant effect of the test item was confirmed by the evaluation of the measured data referring to the ear thickness and number of lymph node cells.

 

The animals of the control and test groups showed a stagnating or declining development of the body weight reflecting the disturbed general state of health of the animals during the study. A positive increase of ear thickness was shown in all three test groups compared to the negative control. The weight determination of ear tissue showed also an increase in all three test group in comparison to the negative control. A slight increase of the weights of the lymph nodes compared to the negative control was only recorded for that test group, which was treated with the 100% test item. An increase of the proliferation was recorded in all three test groups. But the positive threshold value concerning the index of the number of LN cells was passed only in the case of the test groups treated with the test concentrations of 100% and 25%.

Table 1: calculation of means, standard deviations and indices for the increase of the ear thickness, for the weight of the ear tissue and lymph nodes as well as for the number of lymph node cells:

Parameter of investigation

Negative control

Aduct Urea-Sulfuric

100%

50%

25%

Increase of ear thickness

M of ear thickness on day 4 (mm)

0.20

0.23

0.23

0.23

SD of ear thickness on day 4 (% M)

3

6

4

6

Index of ear thickness derived from the comparison of groups on day 4

1.00

1.15

1.12

1.15

Weight of ear

M (g)

0.0097

0.0203

0.0185

0.0194

SD (% M)

7

17

22

10

Index of ear weight

1.00

2.10

1.92

2.01

Weight of lymph nodes

M (g)

0.0152

0.0168

0.0132

0.0137

SD

16

9

30

17

Index of LN weight

1.00

1.10

0.87

0.90

Number of lymph node cells

M

8,913,333

13,166,667

12,226,667

13,026,667

SD (% M)

40

21

40

332

Stimulation index

1.00

1.48

1.37

1.46

M = mean

SD = Standard Deviation

LN = Lymph nodes

Table 2: Calculation of the differentiation index

 

Aduct Urea-Sulfuric

25%

50%

100%

% max. LN cell count index-increase (max. = index 5)

11.5

0.0

11.9

% max. ear thickness increase (max. = index 2)

15.3

11.6

15.3

Differentiation index (DI) = (% max. LN cell count index increase) / (% max. ear thickness increase)

0.75

0.00

0.78

The treatment with the test item in concentrations of 100%, 50% and 25% (v/v) did cause a transgression of the positive threshold value referring to the index of the increase of ear thickness. The index of the number of lymph node cells was also above the positive threshold value in the case of the test concentrations of 100% and 25%.

Differentiation indices lower than 1 were calculated for all three test concentrations indicating the irritation potential of the test item.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information non-specific (irritant) effect
Conclusions:
Based on the results of the study described, a non-specific (irritant) stimulation potential shall be attributed to the test item Aduct Urea-Sulfuric, in the tested concentrations of 25%, 50% and 100%.
Executive summary:

In a dermal sensitization study (OECD 429, GLP) with aduct urea-sulfuric (uronium hydrogen sulphate) in acetone/olive oil (4:1 v/v), young NMRI mices (6 females per dose) were tested using the method of LLNA (IMDS test) at concentrations of 25, 50 and 100% (v/v).

In contrast to the information known on the substance (i.e. no irritation by skin contact in the rabbit, no irritation observed in the acute oral and dermal test) a severe irritant effect of the test item Aduct urea-sulfuric was revealed.

The treatment with the test item in concentrations of 100%, 50% and 25% (v/v) did cause a transgression of the positive threshold value referring to the index of the increase of ear thickness. The index of the number of lymph node cells was also above the positive threshold value in the case of the test concentrations of 100% and 25%.

Differentiation indices lower than 1 were calculated for all three test concentrations indicating the irritation potential of the test item.