Benzenemethanaminium, N,N,N-trimethyl-, 2-hydroxy-2-methylpropanoate (1:1)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2012 to 22 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

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)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

- Test system: Mice, CBA/CaOlaHsd
- Rationale: Recognised as the recommended test system
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age: 1st pre-test: 10-11 weeks (beginning of treatment); 2nd pre-test: 11-12 weeks (beginning of treatment); main study: 10-11 weeks (beginning of treatment)
- Identification: The animals were distributed into the test groups at random and identified by cage number. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiments, animals were identified by cage number.
- Acclimatisation: 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.

Vehicle:

dimethylformamide

Concentration:

1, 2.5, and 5 % (w/w)

No. of animals per dose:

- Number of animals for the pre-tests: 4 females (two for each pre-test)
- Number of animals for the main study: 20 females
- Number of animals per group: 5 females (nulliparous and non-pregnant)
- Number of test groups: 3
- Number of control (vehicle) groups: 1

Details on study design:

FIRST RANGE FINDING TESTS:
- Compound solubility:The highest test item concentration, which could be technically used was a 50% (w/w) solution in dimethylformamide (DMF). Vortexing and warming to 37°C were used to formulate the test item.
- Effect: Systemic toxicity (On the second application day, both treated animals showed severe signs of systemic
toxicity (tumbling, hunchback posture, laboured breathing, reduced spontaneous activity)
and were sacrificed shortly after the second application.)
SECOND RANGE FINDING TESTS:
- Test item concentration: 5 and 10 % (w/w) in dimethylformamide (DMF).
- Irritation: 5%: Erythema score: 1, day 3 and 4; 10%: Erythema score: 1, day 2; score 2, ear swelling and signs of systemic toxicity, day 3; score 1, day 4
CONCLUSION: As the increase in ear thickness did not exceed the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429 at 5% test item concentration, and signs of systemic toxicity were also not observed, 5% was found to be suitable as highest test item concentration.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:

- Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 1, 2.5, and 5% (w/w) in dimethylformamide. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.

- Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytics, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.8 µCi of 3HTdR (equivalent to 79.0 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

- Determination of Incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium (Release, WDT, 30827 Garbsen, Germany). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed. The level of 3HTdR incorporation was then measured on a ß-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

- Determination of Lymph Node Weight and Cell Count:
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 9.3.3 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY®1, Schärfe System, Reutlingen, Germany). The values obtained were taken down manually.

- Determination of Ear Weights:
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

- Interpretation of Raw Data:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal 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.

Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response and the cutoff-value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cutoff-values have been determined using a different strain of mice and can thus not be implicitly adopted.

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). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003). Outliers were not identified.
However, both biological and statistical significance were considered together.

Parameter:

SI

Remarks on result:

other: 1.0% (w/w): 1.22 2.5% (w/w): 1.60 5.0% (w/w): 2.34 However, none of the obtained S.I.s exceeded the threshold value of 3 for a positive response.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 1.0% (w/w): 1070.1 +/- 406.0 2.5% (w/w): 1403.1 +/- 158.3 * 5.0% (w/w): 2054.3 +/- 224.7 * * statistically significant increase vs. control group (p<0.05)

Viability / Mortality:

No deaths occurred during the study period of the main study.

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

Body Weights:

The body weight of the animals, recorded prior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts:

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weight and -cell count was observed in the highest dose group in comparison to the vehicle control group (p<0.05). 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. This threshold was not exceeded in any test item treated group.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A GLP compliant Local Lymph Node Assay was performed with well-characterized test material. Three groups each of five female mice were treated once daily with the test item at concentrations of 1, 2.5, and 5% (w/w) in dimethylformamide by topical application to the dorsum of each ear for three consecutive days. The appropriateness of the used concentrations was previously assessed by two pre-experiments and based on incidences of irritation and severe systemic toxicity at higher doses. A control group of five mice was treated with the vehicle (dimethylformamide) 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 were excised, pooled per animal and immediately weighed. Furthermore, after excision of the lymph nodes, both ears of the mice were punched at the apical area using a biopsy punch and were immediately weighed pooled per animal using an analytical balance. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. Subsequently the suspensions were washed 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.

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, for BALB/c mice, a cutoff-value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

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 1.22, 1.60, and 2.34 were determined with the test item at concentrations of 1, 2.5, and 5.0% (w/w) in dimethylformamide, respectively. A statistically significant increase in DPM value was obtained in the mid and the high dose group in comparison to the vehicle control group (p<0.05) and a dose response was observed. However, none of the obtained S.I.s exceeded the threshold value of 3 for a positive response.

A statistically significant increase in lymph node weight and -cell count was observed in the highest dose group in comparison to the vehicle control group (p<0.05). Still, the cutoff-value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not exceeded in any test item treated group.

Migrated from Short description of key information:
The test item showed no sensitising potential in the LLNA (OECD 429, GLP).

Justification for selection of skin sensitisation endpoint:
GLP and guideline study

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the LLNA, the test item need not to be classified according to Regulation (EC) No 1272/2008 or to Directive 67/548/EEC.