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Administrative data

Description of key information

In a local lymph node assay conducted according to OECD TG 429, a sensitizing potential was not detected.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Guideline 429 and GLP
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/CaOlaHsd
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
Number of animals for the pre-test: 4 females (2 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
Age: 1st pre-test: 9 – 10 weeks (beginning of treatment); 2nd pre-test: 9-10 weeks (beginning of treatment); Main study: 8 - 9 weeks (beginning of treatment)
Identification: The animals were distributed into the test groups at random. 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-experiment, animals were identified by cage number.
Acclimation: 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.
Housing: group
Cage type: Makrolon Type II (pre-test)/III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C, relative humidity approx. 45-65%, artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
dimethylformamide
Concentration:
1, 2 and 5%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% (= highest concentration that could technically be used) once daily each on three consecutive days. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. 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 No systemic toxicity was observed. Test item residues were noted in both animals from day 3 until day 6, while visible ear swelling was observed in both animals on day 6. Moreover, increase in ear thickness was 28.7 and 30.4%, respectively, on day 6. A slight erythema of the ear skin was observed in both animals (score 1, from day 3 until day 4 or 5).
Due to the excessive ear irritation, a second pre-test was performed using test item concentrations of 2 and 5%. A slight crust formation was observed in the animal treated with 5% test item concentration on day 4 to 6. However this was considered to be biologically not relevant, as all other parameters indicating excessive irritation were well within the range recommended by OECD 429 in both animals.
Except for a very slight erythema (score 1) in both animals from day 3 until day 5, no signs of relevant irritation were observed.
Thus, the test item in the main study was assayed at 1, 2, and 5%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY:

TOPICAL APPLICATION:
25 μL/ear/day of the test item were topically (epidermally) applied at concentrations of 1, 2, and 5% in DMF to the entire dorsal surface of each ear for 3 consecutive days. A control group was treated with an equivalent volume of the relevant vehicle alone.
ADMINISTRATION OF 3H-METHYL-THYMIDINE:
5 days after the first topical application 250 μL of phosphate-buffered saline containing 20.2 μCi of 3H-methyl thymidine (equivalent to 80.6 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
DETERMINATION OF INCORPORATED 3HTdR:
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 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CAS®1, Schärfe System). The values obtained were taken down manually.
DETERMINATION OF EAR WEIGHTS:
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
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 the test item groups and negative control group. For all statistical calculations the custom-made statistical program ‘R’ was used. A One-Way-Analysis-of-Variance was used as a 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 detection of possible outliers (performed with Microsoft Excel 2007). However, both biological and statistical significance were considered together.
Parameter:
SI
Remarks on result:
other: See table 1 in section "any other information on results incl. tables".
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table 1 in section "any other information on results incl. tables".

Table 1. Calculation of Stimulation Indices per Dose Group.

 

Test item concentration

Group Calculation

Mean DPM per animal (2 lymph nodes)a)

 

SD

 

S.I.

Vehicle Control Group (DMF)

1423.8

353.1

1.00

1%NM01

1483.2

293.8

1.04

2%NM01

1560.2

348.3

1.10

5%NM01

1883.6

1025.6

1.32

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

Viability/Mortality:

No deaths occurred during the study period.

Clinical Signs:

No signs of systemic toxicity were observed during the study period. Scabby ears were noted in the high dose group on day 4 and 5, but without presence of erythema.

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.

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 or biologically relevant increase in lymph node cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. A statistically significant increase in lymph node weights was observed in the high dose group in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response [Ehling G., Hecht, M., Heusener J., Gamer A.O., van Loveren H., Maurer Th., Riecke K., Ullmann L., Ulrich P., Vandebriel R., Vohr H.-W. (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay 2nd round. Toxicology, 212, 69–79.]. The indices determined for the lymph node cell count did not exceed this threshold.

Ear Weights:

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not exceeded in any of the treated groups.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item NM01 was not a skin sensitiser under the test conditions of this study.
Executive summary:

The skin sensitising potential of NM01 was assessed in a local lymph node assay conducted according to OECD Guideline 429. Groups of five female mice each were treated with the test item at 1, 2, and 5% in DMF by topical application to the dorsum of each ear for three consecutive days.

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, pooled per animal and immediately weighed. 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 pooled lymph nodes 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 β-scintillation counter.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. Scabby ears were noted in the high dose group on day 4 and 5, but without presence of erythema. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group.

In this study Stimulation Indices (S.I.) of 1.04, 1.10 and 1.32 were determined with the test item at concentrations of 1, 2, and 5% in DMF, respectively. A dose response was not observed.

Neither a statistically significant increase in DPM value nor in lymph node cell count was observed in any dose group in comparison to the vehicle control group. A statistically significant increase in lymph node weights was observed in the high dose group in comparison to the vehicle control group. However this was considered not to be biologically relevant since the S.I. of all dose groups were clearly below 3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

As in a local lymph node assay conducted according to OECD TG 429, a sensitizing potential was not detected, the classification criteria of the CLP Regulation are not met.