Reaction mass of Trisodium 4-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate and Trisodium 4-({4-chloro-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate

Endpoint summary

Description of key information

FAT 40000 is not a skin sensitiser.

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:

Experiment start date - 06 June 2016; Experiment completion date - 19 July 2016; Study completion date - 23 August 2016.

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:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Testing Guidelines for Toxicology Studies, 12 NohSan No. 8147, amended 10 December 2002

Version / remarks:

10 December 2002

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:

Code No: FAT 40000/I TE
Physical state/ Appearance: yellow orange powder
Batch No: MC-13
Purity: 71.8%
Expiry date - 27 August 2019
Storage conditions: Approximately - 20 °C in the dark

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes

- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25”C
- Humidity (%): 30-70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 06 June 2016 To: 19 July 2016

Vehicle:

other: 1% pluronic L92 in distilled water

Concentration:

Preliminary Screening Test = 25% w/w in 1% pluronic L92 in distilled water
Main Test = 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water

No. of animals per dose:

Preliminary Screening Test = 1 mouse
Main Test = 4 mice per group

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a concentration of 25% w/w in 1% pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included below. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".

Positive control substance(s):

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

Positive control results:

α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test with a Stimulation Index of 4.47

Key result

Parameter:

SI

Value:

1.25

Test group / Remarks:

5% w/w

Remarks on result:

other: Negative

Key result

Parameter:

SI

Value:

1.65

Test group / Remarks:

10% w/w

Remarks on result:

other: Negative

Key result

Parameter:

SI

Value:

2.33

Test group / Remarks:

25% w/w

Remarks on result:

other: Negative

Cellular proliferation data / Observations:

Preliminary Screening Test
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in 1% pluronic L92 in distilled water.

Main Test
Estimation of the Proliferative Response of Lymph Node Cells
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in
1% pluronic L92 in distilled water Stimulation Index Result
5 1.25 Negative
10 1.65 Negative
25 2.33 Negative

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Clinical observations and mortality data: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body weight: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a "non-sensitizer" under the conditions of the test.

Executive summary:

A study was performed according to OECD test guideline 429 in a GLP certified laboratory, to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in 1% pluronic L92 in distilled water at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in 1% pluronic L92 in distilled water	Stimulation Index	Result
5	1.25	Negative
10	1.65	Negative
25	2.33	Negative

No mortality was observed. Body weight gain was normal during the course of this study.

The test item was considered to be a non-sensitizer under the conditions of the test. The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In total four studies have been conducted to investigate skin sensitization potential of Reactive Yellow 095.

The first two tests conducted in 1990 on batch FAT 40000/G and in 1992 on batch FAT 40000/F followed the testing protocol of the Guinea Pig Maximisation Test according to Magnusson & Kligman. The third test conducted in 2016 on batch FAT 40000/I was done in mice following the protocol of the Local Lymph Node Assay (LLNA). The last test performed in 1994 by Safepharm on “Kayacion Yellow P-7G” is again a study following the Magnusson and Kligman protocol but performed with a non-Huntsman test substance. 

 

Based on the analytical certificates available batch 40000/I consists of equivalent amounts of the meta- and para-isomere of the molecule. The para-isomer is sometimes also described by CAS 111850-26-1 and EC 402-480-0.

 

While the skin sensitization test according to the protocol of the LLNA gave a negative result in mice, the results of the GPM-tests of batch 40000/G and 40000/F gave contradictory results: The first test with batch 40000/F has been judged negative with no animals being sensitized in the test groups, while the second test with batch 40000/G showed a positive result with about 53% of the animals being judged "positive" after the first challenge at a test substance concentration of 25 %. In addition one animal of the test group died for unknown reasons on day 5 of the study. After necropsy no evidence was found that the death was substance related, therefore this result was excluded from the final evaluation and rated as incidental. Beside this further deviations are present within this study. During the epidermal pretest performed with 4 animals the substance was tested at 25, 15, 10 and 5 %. No local effects were noted in all animals for all four concentrations. Therefore, the 25 % concentration was selected as highest non-irritant concentration to be used for the challenge phase but was also used as highest applicable concentration to be used for the induction phase. This was correct.

However, as one can see after the induction phase performed with 25 %, 6 out of 19 test animals showed a skin reaction (edema grade 1), even if the erythema could not be evaluated, this showed that the 25 % concentration produced some effects and it is highly probable that the edema was associated with an erythema (unfortunately not possible to assess as animals are not depilated during the induction phase). The issue is that the same concentration was used during the challenge phase and again some animals showed reactions (erythema + edema in one case). It is not possible to judge if the reaction observed in the animals after challenge is related to irritation or sensitisation, given that some animals showed also effects during the induction phase.

Additionally, when comparing the results at the 24h reading and 48h reading, the number of animals showing effects is reduced at 48h and the animals showing effects at the 48h reading showed less severity than at the 24h reading. This is typical for irritation effects whereas sensitisation effects generally go in the opposite way, more animals after 48h and higher severity.

After the effects observed in the induction phase, the study director should have reduced the concentration to be used for the challenge phase or at least used two different concentrations like 25 and 15 %, as the concentration to be used for a challenge phase in the M&K test should be the highest non-irritant concentration, which is obviously not the case in this study given the edema observed during the induction phase.

In summary this deviation from the guideline in parallel with other uncertainties like great weight loss of one test animal during the acclimatization period, the death of on test group member in the course of the study, the overall reliability of the study is questionable. Consequently, the results of this study should be considered at least as equivocal if not completely invalid.

 

Last but not least two further Guinea Pig Maximization Tests are available.

The first test was conducted on test material FAT 40279/B (representing the pure para-form of the molecule) performed in 1987 according to the test protocol of the GPMT (Magnusson & Kligman) and also gave a negative response with no single animal showing signs of sensitziation, signs of toxicity or death in response to the challenge procedure.

The second test was performed in 1994 by Safepharm on a non-Huntsman test substance according to the test protocol of the GPMT (Magnusson & Kligman) and also gave a negative response with no single animal showing signs of sensitziation, signs of toxicity or death in response to the challenge procedure.

Taking all this information into account the reason why 1/5 tests involving two different test types, two different animal species, test materials from two different companies and different test material compositions regarding the composition of the meta- and para-isomere of the molecule is positive is difficult to judge.

Therefore in a weight of evidence approach the test material is judged not sensitizing to the skin and will therefore not be classified according to GHS.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available