Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl) azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Description of key information

Skin sensitsiation

The test item is not a skin sensitiser under the test conditions of the LLNA study.

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:

2016-03-11 to 2016-04-20

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)

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch no.: 0013479406

Species:

mouse

Strain:

CBA

Remarks:

/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 1st pre-test: 9 - 10 weeks, 2nd pre-test: 11 - 12 weeks, Main study: 10 - 11 weeks
- Weight at study initiation: 19.1 g- 22.1 g
- Housing: Group
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.

Vehicle:

dimethyl sulphoxide

Remarks:

Purity: 99.98 %

Concentration:

1, 2, and 5 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item could be dissolved in the vehicle.
- Irritation: Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin.
- Systemic toxicity: Clinical signs were recorded at least once daily.
- Ear thickness measurements: 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.
Ear irritation was considered if an increase in ear thickness of ≥ 25 % was recorded on day 3 or day 6.
- Erythema scores: 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.

Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 20 % once daily each on three consecutive days.

Result of pre-test:
Both animals showed slight eschar formation upon preparation on day 6. A possible erythema of the ear skin could not be determined, due to the colour of the test item. Therefore, a second pretest was performed using test item concentrations of 2 and 5%. The animals treated with 2 and 5% of the test item showed scaly ears on day 6 but no signs of excessive local skin irritation.
Erythema of the ear skin could not be assessed due to the colour of the test item.

MAIN STUDY

Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: Prior to the first application and prior to treatment with 3HTdR.
Ear weights: In the main experiment after sacrifice; biopsy punches were taken from each ear.
Lymph node weights: In the main experiment, after excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Lymph node cell count: In the main experiment 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): In the main experiment clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Random assignment
- Criteria used to consider a positive response:
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.

TREATMENT PREPARATION AND ADMINISTRATION:
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 1, 2, and 5% in DMSO. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter 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 DMSO alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.6 μCi of 3H-methyl thymidine (equivalent to 78.5 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.

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).
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.
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. No outlier values were detected.
However, both biological and statistical significance were considered together.

Positive control results:

Test group 3 (10 % PC)= SI 2.79
Test group 4 (25 %PC) = SI 7.84
EC3 value = 10.6%

Ear skin irritation: cut-off value of 1.1 for the ear weight index

Key result

Parameter:

SI

Value:

1.02

Variability:

0.59 - 1.47

Test group / Remarks:

1 % test item

Key result

Parameter:

SI

Value:

0.86

Variability:

0.20 - 1.43

Test group / Remarks:

2 % test item

Key result

Parameter:

SI

Value:

1.32

Variability:

0.76 - 2.00

Test group / Remarks:

5 % test item

Cellular proliferation data / Observations:

EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:
Viability / Mortality
No deaths occurred during the study period.

Clinical signs
No signs of systemic toxicity were observed during the study period. On day 6, the animals treated with the low and high dose of the test item and some of the animals treated with the mid dose of the test item showed slightly scaly ears. Moreover, two animals of the mid dose group (animals 14 and 15) and one animal of the high dose group (animal 19) showed slight eschar formation. A possible erythema of the ear skin could not be determined in the mid and high dose group, 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 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. 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 (1.1) 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:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Skin sensitisation

Key study

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice according to OECD 429 and GLP. Test item solutions at different concentrations were prepared in the vehicle DMSO.

Test item concentrations of 1, 2, and 5 % (w/w) were performed. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by two pre-experiments).

The animals neither showed signs of systemic toxicity nor mortality during the course of the study. On day 6, all animals treated with the low dose and high dose of the test item as well as some animals treated with the mid dose of the test item showed slightly scaly ears. Moreover, two animals of the mid dose group (animals 14 and 15) and one animal of the high dose group (animal 19) showed slight eschar formation. A possible erythema of the ear skin could not be examined in the mid and high dose, 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 1.02, 0.86 and 1.32 were determined with the test item at concentrations of 1, 2, and 5 % (w/w) in DMSO, respectively.

A statistically significant or biologically relevant increase in DPM-values, lymph node weights or –cell counts 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 in any treated group.

The test item was thus not a skin sensitiser under the test conditions of this study.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes. In the LLNA the test item was determined to be not a skin sensitiser. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.