Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

Endpoint summary

Description of key information

In an in vivo Local Lymph Node Assay according to OECD Guideline 429, the test item showed skin sensitizing properties (Stahl, 2016).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: experimental data on similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010-07-23

Deviations:

no

GLP compliance:

yes

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: Envigo RMS B.V., Inc.
- Age at study initiation: Pre-test: 8 - 9 weeks; Main study: 10 - 11 weeks
- Weight at study initiation: animals of comparable size and weight
- Housing: group
- Diet: ad libitum
- 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
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

5, 10, 25 %

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 25 % suspension in propylene glycol (PG). Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item could not be achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37 °C).
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 10 and 25 % 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 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.
At the tested concentrations the animals neither showed any sign of local skin irritation nor systemic toxicity. From day 2 to 4, redness of the ear skin could not be assessed due to the colour of the test item.
Thus, the test item in the main study was assayed at 5, 10, and 25 %. 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
Test Item Preparation
The test item was placed into an appropriate container on a tared balance and PG was added. The formulation was sonicated for 10 minutes. The different test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.

Experimental Design and Procedures
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 5, 10, and 25 % in PG. 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 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.5 μCi of 3H-methyl thymidine (equivalent to 82 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 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 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR 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.

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 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 treated 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 and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.

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 experiment after sacrifice; biopsy punches were taken from each ear.
Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Lymph node cell count: The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) 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 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).
The EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with validated program R Script STABW-mitStat.Rnw.
Within the program a statistical analysis 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. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier (DPM value for animal 3) was detected in the Grubb’s, but not in the Dean-Dixon-Test and was therefore not excluded from calculations.
However, both biological and statistical significance were considered together.

Key result

Parameter:

EC3

Remarks:

%

Value:

12.8

Key result

Parameter:

SI

Remarks:

25 %

Value:

4.94

Test group / Remarks:

25 %

Key result

Parameter:

SI

Remarks:

10 %

Value:

2.55

Test group / Remarks:

10 %

Key result

Parameter:

SI

Remarks:

5 %

Value:

1.8

Test group / Remarks:

5 %

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: control: 1033.5 / 5 %: 1861.9 / 10 %: 2637.3 / 25 %: 5107.3

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity were observed during the study period. Redness of the ear skin could not be assessed, due to the colour of the test item.

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 and biologically relevant increase in lymph node weights was observed in all 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. The indices determined for the lymph node cell count of the high dose group exceeded this threshold (indexs of 1.68).

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:

other: classified as H317 Cat 1B under Regulation 1272/2008

Conclusions:

Skin sensitizer

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. The study was conducted according to OECD 429 guideline and GLP (Stahl, 2016). Test item suspensions at different concentrations were prepared in the vehicle PG (propylene glycol). 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 5, 10, and 25 % (w/w). The highest concentration tested was the highest concentration that could be technically achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

Three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25 % in propylene glycol (PG) by topical application to the dorsum of each ear for three consecutive days. A control group of five mice was treated with the vehicle PG 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 neither showed any signs of systemic toxicity nor mortality during the course of the study. Redness of the ear skin could not be assessed, due to the colour of the test item. 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 1.80, 2.55, and 4.94 were determined with the test item at concentrations of 5, 10, and 25 % (w/w) in PG, respectively. A clear dose response was observed. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight was observed in all dose groups as well as in the high dose group for the lymph node cell count 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 exceeded in the high dose group (index of 1.68).

In conclusion, the test item was found to be a skin sensitizer and an EC3 value of 12.8 % was derived.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As a result the test substance should be considered to be classified as skin sensitiser (UN GHS: Category 1B, H317) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

Based on the read across considerations the target substance is classified as Category 1B, H317