Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-04 to 2013-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Golden Yellow HF-RN 1331

IUPAC Name:

Reactive Golden Yellow HF-RN 1331

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: Reactive Golden Yellow HF-RN 1331
Batch No.: Kilo 10&12
Chemical Name: 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4 pyrimidinyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt
Formula: C17H11F2N7Na2O7S2
Physical State: solid (powder)
Colour: reddish orange
pH: 4.5 at 5%
Molecular Weight: 573.42 g/mol
Purity: 100 % (composition see AZ. No. 12-1327/2473 v.2, dated 2012-10-08)
Storage Conditions: room temperature
Expiry Date: 17.01.2014
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

- Full barrier in an air-conditioned room
-Temperature: 22 ± 3 °C
-Relative humidity: 55 ± 10%
-Artificial light, sequence being 12 hours light, 12 hours dark
-Air change: at least 10 x / hour
-Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test and main study: lot no. 0902)
-Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
-The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
(prescreen test: lot no. 011012, main study: lot no. 240113)
-Certificates of food, water and bedding are filed at BSL BIOSERVICE
-Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:

other: 2% carboxymethylcellulose (CMC) in aqua ad injectionem

Concentration:

Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
3%, 6% and 12.5% (w/v)

No. of animals per dose:

5 animals/dose group, 5 animals/control group

Details on study design:

Prescreen Test

Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item in the vehicle was found to be 12.5% in 2% CMC in aqua ad injectionem.
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted
under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any
clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination.
Both ears were observed for erythema and scored. Ear thickness measurements were performed on day 1 (pre-dose),
day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear
swelling of ≥ 25%.
Two animals were treated by topical application with the test item on three consecutive days at a concentration of 12.5% (dissolved in 2% CMC)
to the entire dorsal surface of each ear.
One further animal was treated with 100% CMC (undiluted) and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured.
During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal.
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test.

Main Study

Preparation of the Animals
The animals were randomly selected.
Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).
Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application.
Topical applications were performed once daily over three consecutive days.
Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of
250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. Shortly before sacrificing
the thickness of the ears of all animals was measured for a third time. The draining auricular lymph nodes were excised, individually pooled
for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell
suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure
was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of
macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H-Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM).
Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually
for each animal.


Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node
(DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for
control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation
{EC3=c+[(3-d)/(b-d)]x(a c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the
stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in
3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of
control group animals (Stimulation Index equal to or greater than

Positive control substance(s):

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

Statistics:

Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM). If outliers were identified, these values were not included in the calculation of the stimulation indices. As at least four values per group are required for the evaluation of the results, the outlier test was not repeated to detect further outliers.

Results and discussion

Positive control results:

One of the three tested concentrations exceeded the stimulation index of 3.
The stimulation index at a concentration of 3% was 2.1
The stimulation index at a concentration of 6% was 2.3
The stimulation index at a concentration of 12.5% was 6.1
The stimulation index of the positive control (25% α-Hexylcinnamaldehyde in 2% CMC in aqua ad injectionem) was 9.1 and therefore the test
is considered to be valid.

All animals survived throughout the test period without showing any clinical signs.

Body Weight Development
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Measurement of Ear Thickness
Directly prior to the first application, approximately 48 hours after the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured.
The means of the ear thickness per group showed no relevant difference compared to the negative control.
The mean ear thickness on day 1 day 3 day 6
for the 3% test group was 0.20 mm 0.20 mm 0.19 mm
for the 6% test group was 0.19 mm 0.20 mm 0.19 mm
for the 12.5% test group was 0.19 mm 0.20 mm 0.20 mm
for the negative control group was 0.19 mm 0.19 mm 0.19 mm
for the positive control group was 0.19 mm 0.21 mm 0.21 mm

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

One of the three tested concentrations exceeded the stimulation index of 3. The stimulation index at a concentration of 3% was 2.1 The stimulation index at a concentration of 6% was 2.3 The stimulation index at a concentration of 12.5% was 6.1 The stimulation index of the positive control (25% α-Hexylcinnamaldehyde in 2% CMC in aqua ad injectionem) was 9.1 and therefore the test is considered to be valid.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Animal number DPM DPM- mean back- ground 16 1187.0 1166.0 583.0 17 1670.0 1649.0 824.5 18 659.0 638.0 319.0 19 523.0 502.0 251.0 20 651.0 630.0 315.0 MV 938.0 917.0 458.5 SD 431.3 431.3 215.7 21 7557.0 7536.0 3768.0 22 5424.0 5403.0 2701.5 23 10658.0 10637.0 5318.5 24 11852.0 11831.0 5915.5 25 6354.0 6333.0 3166.5 MV 8369.0 8348.0 4174.0 SD 2480.5 2480.5 1240.2 1 2670.0 2649.0 1324.5 2 5315.0* n.d. n.d. 3 1527.0 1506.0 753.0 4 1859.0 1838.0 919.0 5 1597.0 1576.0 788.0 MV 1913.3 1892.3 946.1 SD 454.1 454.1 227.0 6 579.0 558.0 279.0 7 3918.0 3897.0 1948.5 8 3609.0 3588.0 1794.0 9 1610.0 1589.0 794.5 10 1094.0 1073.0 536.5 MV 2162.0 2141.0 1070.5 SD 1351.2 1351.2 675.6 11 2395.0 2374.0 1187.0 12 5570.0 5549.0 2774.5 13 4306.0 4285.0 2142.5 14 6848.0 6827.0 3413.5 15 9099.0 9078.0 4539.0 MV 5643.6 5622.6 2811.3 SD 2268.8 2268.8 1134.4 22.0 18.0 24.0 19.0 22.0 MV 21.0 0.0 0.0 SD 2.2 * = outlier, failed Grubbs, Nalimov and Dixon; n.d. = not determined

Any other information on results incl. tables

Table Ear Thickness – Preliminary Test (mm)

	Animal No.	Measurement of Ear Thickness (mm)
Concentration		Day 1		Day 3		Day 6
		left	right	left	right	left	right
Reactive Golden Yellow HF-RN 1331 12.5% in CMC (2%)	1	0.17	0.17	0.18	0.17	0.18	0.18
Reactive Golden Yellow HF-RN 1331 12.5% in CMC (2%)	2	0.18	0.17	0.18	0.17	0.17	0.17
Negative Control 100% CMC (2%)	3	0.18	0.18	0.17	0.17	0.17	0.17

Table Absolute Body Weights – Preliminary Test (g)

Concentration	Animal No.	Start of Study	End of Study	Weight Gain
Reactive Golden Yellow HF-RN 1331 12.5% in CMC (2%)	1	23	23	0
Reactive Golden Yellow HF-RN 1331 12.5% in CMC (2%)	2	22	21	-1
Negative Control 100% CMC (2%)	3	22	23	1

Table Radioactive Determination of the Test Substance and Control Groups

POS	CPM			Test Item	Conc. [%]	Animal number	DPM	DPM- mean back- ground	DPM/ Node	Stimu-lation Index
56	571.0			Negative	100	16	1187.0	1166.0	583.0
57	805.0			Control		17	1670.0	1649.0	824.5
58	319.0			CMC (2 %)		18	659.0	638.0	319.0
59	253.0					19	523.0	502.0	251.0
60	311.0					20	651.0	630.0	315.0
MV	451.8					MV	938.0	917.0	458.5	1.0
SD	207.8					SD	431.3	431.3	215.7
61	3649.0			Positive	25	21	7557.0	7536.0	3768.0	8.2
62	2545.0			Control		22	5424.0	5403.0	2701.5	5.9
63	5105.0			α-Hexylcinnamaldehyde		23	10658.0	10637.0	5318.5	11.6
64	5652.0			in CMC (2 %)		24	11852.0	11831.0	5915.5	12.9
65	3066.0					25	6354.0	6333.0	3166.5	6.9
MV	4003.4					MV	8369.0	8348.0	4174.0	9.1
SD	1188.5					SD	2480.5	2480.5	1240.2	2.7
66	1244.0			Reactive Golden	3	1	2670.0	2649.0	1324.5	2.9
67	2566.0*			Yellow HF-RN 1331		2	5315.0*	n.d.	n.d.	n.d.
68	733.0			in CMC (2 %)		3	1527.0	1506.0	753.0	1.6
69	894.0					4	1859.0	1838.0	919.0	2.0
70	768.0					5	1597.0	1576.0	788.0	1.7
MV	909.8					MV	1913.3	1892.3	946.1	2.1
SD	202.1					SD	454.1	454.1	227.0	0.5
71	281.0			Reactive Golden	6	6	579.0	558.0	279.0	0.6
72	1873.0			Yellow HF-RN 1331		7	3918.0	3897.0	1948.5	4.2
73	1757.0			in CMC (2 %)		8	3609.0	3588.0	1794.0	3.9
74	775.0					9	1610.0	1589.0	794.5	1.7
75	529.0					10	1094.0	1073.0	536.5	1.2
MV	1043.0					MV	2162.0	2141.0	1070.5	2.3
SD	650.4					SD	1351.2	1351.2	675.6	1.5
76	1148.0			Reactive Golden	12.5	11	2395.0	2374.0	1187.0	2.6
77	2683.0			Yellow HF-RN 1331		12	5570.0	5549.0	2774.5	6.1
78	2042.0			in CMC (2 %)		13	4306.0	4285.0	2142.5	4.7
79	3302.0					14	6848.0	6827.0	3413.5	7.4
80	4367.0					15	9099.0	9078.0	4539.0	9.9
MV	2708.4					MV	5643.6	5622.6	2811.3	6.1
SD	1093.9					SD	2268.8	2268.8	1134.4	2.5
81	11.0			Background			22.0
82	9.0			Szinti and			18.0
83	12.0			TCA			24.0
84	9.0						19.0
85	11.0						22.0
MV	10.4					MV	21.0	0.0	0.0	0.0
SD	1.2					SD	2.2
* = outlier, failed Grubbs, Nalimov and Dixon;   n.d. = not determined

Table Absolute Body Weights in g

Concentration	Animal No.	Start of Study	End of Study	Weight Gain
	1	21	22	1
Reactive Golden	2	22	23	1
Yellow HF-RN 1331	3	19	20	1
3 % in CMC (2%)	4	19	21	2
	5	18	21	3
	6	21	21	0
Reactive Golden	7	19	20	1
Yellow HF-RN 1331	8	21	22	1
6 % in CMC (2%)	9	21	23	2
	10	20	21	1
	11	19	21	2
Reactive Golden	12	18	20	2
Yellow HF-RN 1331	13	20	23	3
12.5 % in CMC (2%)	14	21	23	2
	15	20	21	1
	16	20	22	2
Negative Control	17	19	20	1
100% CMC (2%)	18	20	22	2
	19	18	21	3
	20	21	23	2
Positive Control 25% α-Hexyl- cinnamaldehyde in CMC (2%)	21	22	23	1
	22	19	21	2
	23	21	22	1
	24	20	20	0
	25	19	20	1

Table Clinical Observation

Time of Observation	Systemic Effects	Local Effects
Group 1, animals no. 1 – 5 / test item at a concentration of 3% inin CMC (2%)
Day 1	nsf	nsf
Day2	nsf	nsf
Day3	nsf	nsf
Day4	nsf	nsf
Day5	nsf	nsf
Day6	nsf	nsf
Group 2, animals no. 6 – 10 / test item at a concentration of 6%in CMC (2%)
Day 1	nsf	nsf
Day2	nsf	nsf
Day3	nsf	nsf
Day4	nsf	nsf
Day5	nsf	nsf
Day6	nsf	nsf
Group 3, animals no. 11 – 15 / test item at a concentration of 12.5%in CMC (2%)
Day 1	nsf	nsf
Day2	nsf	nsf
Day3	nsf	nsf
Day4	nsf	nsf
Day5	nsf	nsf
Day6	nsf	nsf
Group 4, animals no. 16 – 20 / negative control100% CMC (2%)
Day 1	nsf	nsf
Day2	nsf	nsf
Day3	nsf	nsf
Day4	nsf	nsf
Day5	nsf	nsf
Day6	nsf	nsf
Group 5, animals no. 21 – 25 / positive control 25%in CMC (2%)
Day 1	nsf	nsf
Day2	nsf	nsf
Day3	nsf	nsf
Day4	nsf	nsf
Day5	nsf	nsf
Day6	nsf	nsf

nsf = no specific findings

Applicant's summary and conclusion

Conclusions:

The most conservative EC3 value (= estimated concentration needed to produce a stimulation index of 3, derived by linear interpolation) was
calculated to be at a test item of 7.14%.
Under the conditions of this study the test item Reactive Golden Yellow HF-RN 1331 was found to be a skin sensitizer.
The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group.

Executive summary:

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 3%, 6% and 12.5% (w/v), each diluted with 2% carboxymethylcellulose (CMC) inaqua ad injectionem. The concentration of 12.5% in 2% CMC in aqua ad injectionem was the maximum technically applicable concentration of the test item in the vehicle.

At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed.Species/strain:                     Mice, CBA/CaOlaHsd

Number of animals:            25/main test, 3/prescreen test

Vehicle:                               2% carboxymethylcellulose (CMC) inaqua ad injectionem.

Summary Results

One of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration          of       3%      was     2.1

The stimulation index at a concentration          of       6%      was     2.3

The stimulation index at a concentration          of       12.5% was     6.1

 

The mean ear thickness on	day 1	day 3	day 6
for the 3% test group was	0.20 mm	0.20 mm	0.19 mm
for the 6% test group was	0.19 mm	0.20 mm	0.19 mm
for the 12.5% test group was	0.19 mm	0.20 mm	0.20 mm
for the negative control group was	0.19 mm	0.19 mm	0.19 mm
for the positive control group was	0.19 mm	0.21 mm	0.21 mm

The stimulation index of the positive control (25% α-Hexylcinnamaldehyde in2% CMC in aqua ad injectionem) was 9.1 and therefore the test is considered to be valid.

 Conclusion

The most conservative EC3 value (= estimated concentration needed to produce a stimulation index of 3, derived by linear interpolation) was calculated to be at a test item of 7.14%.

Under the conditions of this study the test item Reactive Golden Yellow HF-RN 1331 was found to be a skin sensitizer.

The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group.