Methyl hydrogen 2-nitroterephthalate

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: Well performed GLP and OECD 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)

Version / remarks:

reduced LLNA approach

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl hydrogen 2-nitroterephthalate

EC Number:

252-360-5

EC Name:

Methyl hydrogen 2-nitroterephthalate

Cas Number:

35092-89-8

Molecular formula:

C9H7NO6

IUPAC Name:

4-(methoxycarbonyl)-3-nitrobenzoic acid

Details on test material:

- Name of test material (as cited in study report): Nitro-MMT TRTR

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

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: 18.1 - 21.4 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), 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): 22 +/- 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

Test item concentration: 25% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50 % (w/v) solution in dimethylsulfoxide. Vortexing was necessary to formulate the test item.
To determine the highest non-irritant test concentration that did at the same time not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50 % once daily each on three consecutive days. 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. Eventual signs of local skin irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, 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). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema 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 (for individual results see Annex 1). At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. However, at 50% test item concentration, an increase in ear weight was observed that exceeded the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429. The measured ear thickness was also distinctly increased in comparison to the measurement on day 1 prior to the first application of the test item.
The test item in the main study was thus assayed at 25% test item concentration. Since no relevant exposure is expected for this intermediate and since a negative prediction is made based on test results of substances with similar chemical structure (such as Nitro-MMT-dichloroanilide, 83929-47-9) the rLLNA protocol (reduced LLNA approach) was considered to be appropriate.

MAIN STUDY:

TOPICAL APPLICATION:
The test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with a test item concentration of 25 % (w/w) in dimethylsulfoxide. The application volume, 25 µL was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A futher 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 intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). 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 trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation 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 sensitiliser 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: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test and main test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for body weight and for the DPM values.
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). Outliers were not determined.
Also, the ANOVA (Dunnett-test) was conducted on the ear weights to assess whether the difference was statistically significant between the test item group and negative control (vehicle) group. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

Experiment performed in December 2011 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I. values of 1.70, 1.81, and 5.90, respectively.
The EC3 value calculated was 14.4 % (w/v).
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)

Results

Results 1

Parameter:

SI

Remarks on result:

other: S.I. Vehicle Control Group: 1.00 25 % Nitro-MMT TRTR: 1.18

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM per animal Vehicle Control Group: 763.1 25 % Nitro-MMT TRTR: 903.5

Any other information on results incl. tables

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No signs of systemic toxicity were observed during the study period. On day 4 and 5, the test item treated animals showed an erythema of the ear skin (Score 1).

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 WEIGHT

The measured ear weight of all animals treated was recorded on day 6 after necropsy. A statistically significant and biologically relevant increase in ear weight was observed in the test group in comparison to the vehicle control group (p<0.05).

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The test item Nitro-MMT TRTR was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item Nitro-MMT TRTR dissolved in dimethylsulfoxide was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay according to OECD 429 was performed using a test item concentration of 25 %. The concentration tested was the highest concentration that could be applied whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a preexperiment. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 4 and 5, the test item treated animals showed an erythema of the ear skin (Score 1). Furthermore, a statistically significant and biologically relevant increase in ear weight was observed in the test group in comparison to the vehicle control group. In this study a Stimulation Index (S.I.) of 1.18 was determined with the test item at a concentration of 25 % in dimethylsulfoxide. A statistically significant increase in comparison to the vehicle control group was not observed. The test item Nitro-MMT TRTR was thus not a skin sensitiser under the test conditions of this study.