Registration Dossier

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: well performed GLP and OECD guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,5-Dimethoxynitrobenzol

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Test animals: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16.5 - 23.4 g
- Housing: group housing
- Diet: pelleted standard diet, ad libidum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24°C
- Humidity (%): 35-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: graunulated soft wood bedding

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The highest test item concentration, which can be technically used was a 50% (w/v) solution in acetone:olive oil (4+1) after sonicating and warming at 37°C.
In the pre-test, two mice were treated with test item concentrations of 25 and 50%.
The test item in the main study was assayed at 10, 25 and 50%.
No. of animals per dose:
4
Details on study design:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with test item concentrations of 10, 25, and 50% (w/v) in acetone:olive oil (4:1). The application volume, 25 µL, was spread over the entire dorsal surface (diameter ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF ³H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED ³H-METHYL THYMIDINE:

Five days after the first topical application, all mice were administered with 250 µL of 81.5 µCi/ml 3HTdR (corresponds to 20.4 µCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after treatment with ³HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA:

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that 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.
- Second, that 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.

OBSERVATIONS:

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: Once daily (week day) from experimental start to necropsy.
- Body weights:Prior to the first application and prior to treatment with 3HTdR.
- Ear weights: After sacrifice. Biopsy punches were taken from each ear.
- Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour after each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For body weight mean values and standard deviations were calculated.
The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between ear weights of test item groups and the negative control (vehicle) group. Statistical significance was 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 experiment with the positive control substance was performend in January 2009 , using concentrations of 5, 10, and 25% alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1 v/v). Stimulation Indices of 1.49, 4.17, and 4.90, were determined. The EC3 value was calculated to be 7.8%.
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation Indices 10%: 1.43 25%: 1.54 50%: 1.43
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per lymph node Control: 669.6 10%: 734.4 25%: 792.6 50%: 737.8

Any other information on results incl. tables

Calculation and results of individual data; Vehicle: acetone/olive oil (4:1 v/v)

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

117

---

---

---

---

---

BG II

32

---

---

---

---

---

1

4195

4121

8

515.1

---

10

2

5950

5876

8

734.4

1.43

25

3

6415

6341

8

792.6

1.54

50

4

5977

5903

8

737.8

1.43

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1    = Control Group

2-4= Test Group

S.I. = Stimulation Index

a)   = The mean value was taken from the figures BG I and BG II

b)    = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.'s are below 3.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

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.

EAR WEIGHTS

A significant increase in ear weights was observed in all treatment groups compared with the control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item 2,5-Dimethoxynitrobenzol was not a skin sensitizer under the test conditions of this study.
Executive summary:

In this study the test item 2,5-Dimethoxynitrobenzol dissolved in acetone/olive oil (4:1 v/v) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay according to OECD guideline 429 was performed using test item concentrations of 10, 25, and 50%. A control group of four mice was treated with the vehicle (acetone:olive oil (4 +1)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloracetic acid overnight. The proliverative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a beta-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test 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 concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.43, 1.54, and 1.43 were determined with the test item at concentrations of 10, 25, and 50% (w/v) in acetone/olive oil (4:1 v/v). The EC3 value was not calculated since none of the tested concentrations induced an S.I. greater than 3. A significant increase in the ear punch biopsies was observed in all treatment groups compared with the control group. However, the data showed that the test item did not induce a relevant proliferation in the draining lymph nodes, thus the observed increase was not attributed to a sensitising effect. In conclusion, the test item 2,5 -Dimethoxynitrobenzol was not a skin sensitiser under the described conditions.