4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Endpoint summary

Description of key information

Disperse Red 092 was considered to be a non-sensitizer.

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:

03 August 2016 to 01 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

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:

EC 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PCR92X140707 (China)
- Expiration date of the lot/batch: 14 October 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °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 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h darkness
- IN-LIFE DATES: From: 03 August 2016 To: 01 September 2016

Vehicle:

dimethylformamide

Concentration:

Preliminary Screening Test = 50 % w/w
Main Test = 50, 25 and 10 % w/w

No. of animals per dose:

Preliminary ScreeningTest = 1
Mani Test = 4

Details on study design:

Preliminary Screening Test
Using available information 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 50 % w/w in dimethyl formamide, 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 recognised scale (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 50 %, 25 % or 10 % w/w in dimethyl formamide. 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 gamma-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).

Scale for Erythema
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar
formation preventing grading of erythema 4

Positive control substance(s):

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

Positive control results:

A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitizer. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.
Test item: α Hexylcinnamaldehyde, tech., 85 %
Study number: 41502435
Study dates: 04 November 2015 to 10 November 2015

Methods
A group of five animals was treated with 50 µL (25 µL per ear) of α Hexylcinnamaldehyde, tech., 85 % as a solution in dimethyl formamide at a concentration of 25 % v/v. A further control group of five animals was treated with dimethyl formamide alone.

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

Concentration (% v/v) in
dimethyl formamide Stimulation Index Result
25 6.08 Positive

Conclusion
α Hexylcinnamaldehyde, tech., 85 % was considered to be a sensitizer under the conditions of the test.

Key result

Parameter:

SI

Value:

1.16

Test group / Remarks:

10 % w/w

Remarks on result:

other: Negative

Key result

Parameter:

SI

Value:

1.34

Test group / Remarks:

25 % w/w

Remarks on result:

other: Negative

Key result

Parameter:

SI

Value:

1.03

Test group / Remarks:

50 % w/w

Remarks on result:

other: Negative

Cellular proliferation data / Observations:

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 dimethyl formamide Stimulation Index Result
10 1.16 Negative
25 1.34 Negative
50 1.03 Negative

Clinical Observations and Mortality Data:
Red colored staining on the ears was noted in all test animals post dose on Days 1 to 3. The staining did not prevent accurate evaluation of erythema. 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.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 40444/B was considered to be a non-sensitizer under the conditions of the test.

Executive summary:

A study was performed 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. The study was carried out according to OECD guideline 429 and EU method B.42. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 % 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 dimethyl formamide at concentrations of 50 %, 25 % or 10 % w/w. A further group of four animals was treated with dimethyl formamide alone. Red colored staining on the ears was noted in all test animals post dose on Days 1 to 3.  The staining did not prevent accurate evaluation of erythema. There were no deaths.  No signs of systemic toxicity were noted in the test or control animals during the test. 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 dimethyl formamide	Stimulation Index	Result
10	1.16	Negative
25	1.34	Negative
50	1.03	Negative

 

Based on the study results, FAT 40444/B was considered to be a non-sensitizer under the conditions of the test and 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:

A study was performed 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. The study was carried out according to OECD guideline 429 and EU method B.42. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 % 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 dimethyl formamide at concentrations of 50 %, 25 % or 10 % w/w. A further group of four animals was treated with dimethyl formamide alone. Red colored staining on the ears was noted in all test animals post dose on Days 1 to 3. The staining did not prevent accurate evaluation of erythema. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. The Stimulation Indices (expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group) were below 3 for all the treatment groups. Hence, FAT 40444/B was considered to be a non-sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of a local lymph node assay, Disperse Red 092 should not be classified as Skin Sensitiser according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.