Anthranilamide

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

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 ANIMALS
- Source: Harlan Laboratories B.V, Netherlands
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 9 - 12 weeks
- Weight at study initiation: 19 - 22 g
- Housing: Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light):12/12

Vehicle:

dimethyl sulphoxide

Concentration:

0.1, 0.25, and 0.5%

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 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% suspension in DMSO. At a concentration of 25% and below, the test item could be dissolved in the vehicle. Vortexing was used to formulate the test item
- Irritation: Vortexing was used to formulate the test item. To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. 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. Clinicalsigns were recorded at least once daily. Eventual signs of local 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. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (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

MAIN STUDY:
- Topical application:Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.1, 0.25, and 0.5% in DMSO. 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 (control animals).
- Administration of 3 H-methyl-thymidine: Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3 H-methyl thymidine (equivalent to 79.3 µCi/mL 3 HTdR) were injected into each test and control mouse via the tail vein.
- Determination of incorporated 3 HTdR: Approximately five hours after treatment with 3 HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) 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 scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3 HTdR incorporation was then measured in a βscintillation counter. Similarly, background 3 HTdR levels were also measured in two 1 mLaliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3 HTdR incorporation as the number of radioactive disintegrations per minute.
- 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 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY® DT, 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and DMSO was added. The different test item concentrations were prepared individually. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.

Positive control substance(s):

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

Parameter:

SI

Value:

1

Test group / Remarks:

0%

Remarks on result:

other: negative control

Key result

Parameter:

SI

Value:

1.21

Test group / Remarks:

0,50%

Parameter:

SI

Value:

1.31

Test group / Remarks:

0,25%

Parameter:

SI

Value:

1.18

Test group / Remarks:

0,10%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Concentration Disintegrations per minute (DPM)
0% 2070.7
0,10% 2440.5
0,25% 2710.1
0,50% 2509.1

Viability / Mortality: No deaths occurred during the study period.
Clinical Signs: No signs of systemic toxicity were observed during the study period. From day 3 to 5, the animals treated with 0.25 and 0.5%, showed an erythema of the ear skin.
Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with 3 HTdR, was within the range, commonly recorded for animals of this strain and age. However slight weight loss in the control group was observed (-3.6%).

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 weights or cell counts was not observed in any of the test item treated groups 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 (See Ref. 8). The indices determined for the lymph node cell count did not reach or 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 of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item Anthranilamide was not a skin sensitizer.

Executive summary:

In order to study a possible skin sensitising potential of Anthranilamide, three groups each of five female mice were treated once daily with the test item at concentrations of 0.1, 0.25, and 0.5% in DMSO by topical application to the dorsum of each ear for three consecutive days. The test item could be dissolved in the vehicle. The appropriateness of the used concentrations was previously assessed by four pre-experiments. A control group of five mice was treated with the vehicle DMSO 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, 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 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 showed neither signs of systemic toxicity nor mortality during the course of the study. From day 3 to 5, the animals treated with 0.25 and 0.5% showed an erythema of the ear skin (score 1). 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 cut-off 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 reached or 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 1.18, 1.31, and 1.21 were determined with the test item at concentrations of 0.1, 0.25, and 0.5% (w/w) in DMSO, respectively. A statistically significant or biologically relevant increase in DPM value and also in lymph node weight and - cell count was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In this study the test item Anthranilamide was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle DMSO. The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations. For this purpose a local lymph node assay was performed using test item concentrations of 0.1, 0.25, and 0.5% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined four pre-experiments). The animals showed neither signs of systemic toxicity nor mortality during the course of the study. From day 3 to 5, the animals treated with 0.25 and 0.5% showed an erythema of the ear skin (score 1). 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 cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation (see Ref. 9). None of the indices determined for the test item treated groups reached or 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 1.18, 1.31, and 1.21 were determined with the test item at concentrations of 0.1, 0.25, and 0.5% (w/w) in DMSO, respectively. A statistically significant or biologically relevant increase in DPM value and also in lymph node weight and – cell count was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice (see Ref. 8) was not reached or exceeded in any dose group. The test item Anthranilamide was thus not a skin sensitiser under the test conditions of this study.

Migrated from Short description of key information:
The substance is considered to be not skin sensitizing.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the absence of effects after skin exposure, classification for skin sensitization is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008. Furthermore, no classification and labeling is needed in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.