Heptasodium bis[4-hydroxy-5-[(2-hydroxy-1-naphthyl)azo]-3-[(2-hydroxy-3-nitro-5-sulphophenyl)azo]naphthalene-2,7-disulphonato(5-)]cobaltate(7-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 18. Feb. to 04. Mar. 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read-across justification is detailed in section 13; source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

yes

Remarks:

The recording of the climate conditions in the animal rooms was missed out for several hours during the pre-experiment.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V.
- Age at study initiation: Pre-test and main study: 10 - 11 weeks
- Weight at study initiation: animals of comparable size and weight
- Housing: group housing
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 45-65 %
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1 %, 2 % and 5 %

No. of animals per dose:

5

Details on study design:

Vehicle and dose selection
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 10 % suspension in DMF. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing).
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 5 and 10 % 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 did not show any signs of systemic toxicity. A possible erythema of the ear skin could not be assessed due to the inherent colour of the test item.
The animal treated with the high dose concentration (10 %) showed excessive irritation of the ear skin, as indicated by an increase in ear thickness by 25.8 % on day 6 of treatment. The animal treated with 5 % test item concentration did not show any signs of excessive ear skin irritation.
Thus, the test item in the main study was assayed at 1, 2, and 5 %. 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.

Test item preparation
The test item was placed into an appropriate container on a tared balance and DMF was added.
The different test item concentrations were prepared individually. Homogeneity of the test item in 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.

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 DMF. The application volume, 25 μl ear/day, was spread over the entire dorsal surface (Ø 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 20.3 μCi of 3H-methyl thymidine (equivalent to 81.0 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).
A statistical analysis was 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. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree).

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

control

Results 2

Parameter:

SI

Value:

1.27

Test group / Remarks:

1 %

Results 3

Parameter:

SI

Value:

2.01

Test group / Remarks:

2 %

Results 4

Parameter:

SI

Value:

2.22

Test group / Remarks:

5 %

Results 5

Parameter:

other: disintegrations per minute (DPM)

Value:

1 771

Test group / Remarks:

control

Results 6

Parameter:

other: disintegrations per minute (DPM)

Value:

2 248.6

Test group / Remarks:

1 %

Results 7

Parameter:

other: disintegrations per minute (DPM)

Value:

3 553.4

Test group / Remarks:

2 %

Results 8

Parameter:

other: disintegrations per minute (DPM)

Value:

3 935

Test group / Remarks:

5 %

Any other information on results incl. tables

- Viability / mortality: no deaths occurred during the study period.

- Clinical signs: no signs of systemic toxicity were observed during the study period; a possible erythema of the ear skin could not be assessed, due to the inherent 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 was not observed in any of the test item treated groups in comparison to the vehicle control group. A statistically significant increase in lymph node cell counts was observed in the animals of the mid and high dose concentration, respectively. However, 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.

Table 1: Calculation and results of individual data.

Test item concentration			DPM values measured	DPM−BG per animal (2 lymph nodes)^a)	S.I.^b)
% (w/w)	Group no.	Animal no.
-	-	BG I	21	-	-
-	-	BG II	31	-	-
0	1	1	2724	2698	-
		2	1005	979	-
		3	1587	1561	-
		4	1909	1883	-
		5	1760	1734	-
1	2	6	2104	2078	1.2
		7	2248	2222	1.3
		8	1947	1921	1.1
		9	3206	3180	1.8
		10	1868	1842	1.0
2	3	11	3569	3543	2.0
		12	3083	3057	1.7
		13	4554	4528	2.6
		14	2738	2712	1.5
		15	3953	3927	2.2
5	4	16	1476	1450	0.8
		17	5265	5239	3.0
		18	4507	4481	2.5
		19	4116	4090	2.3
		20	4441	4415	2.5

1 = Control Group

2-4 = Test Group

a) = values corrected for mean background value (BGI and BGII)

b) = Stimulation Indices relative to the mean of the control group (Group 1)

Table 2: Calculation of stimulation indices per dose group.

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	SI
Vehicle control group (DMF)	1771.0	621.4	1.00
1 % test item	2248.6	540.8	1.27
2 % test item	3553.4	714.8	2.01
5 % test item	3935.00	1451.6	2.22

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals).

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to the CLP Regulation (EC) no. 1272/2008

Conclusions:

The test item was not found to be a skin sensitiser under the test conditions.

Executive summary:

The skin sensitising potential of the test item was evaluated in an experimental study according to the OECD Guideline 429 (2010). three groups each of five female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine ( 3 H-methyl thymidine; 3 HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed using an analytical balance. Furthermore, both ears of mice were punched at the apical area using a biopsy punch and the punches were immediately weighed pooled per animal using an analytical balance. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled 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 then determined by the incorporation of 3 H-methyl thymidine measured in a βscintillation counter.

No mortality or signs of systemic toxicity were observed during the study period. A possible erythema of the ear skin could not be assessed in all dose groups due to the inherent colour of the test item. A statistically significant 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. In this study, Stimulation Indices (S.I.) of 1.27, 2.01 and 2.22 were determined with the test item at concentrations of 1, 2, and 5 % in DMF, respectively. A dose-response relationship was observed. An outlier was identified in both statistical outlier tests in the group treated with 1 % test item concentration (DPM value determined for animal number 9). Another outlier was identified in the Grubb`s test only (DPM value determined for animal number 16). However, as exclusion of these outliers did not change the overall test result, the values in question were not excluded from any subsequent calculations. A statistically significant or biologically relevant increase in DPM values was not observed in any treated group in comparison to the vehicle control group. A statistically significant increase in lymph node weights was observed in the low dose group in comparison to the vehicle control group. This increase is most likely attributed to the data not being normally distributed. A statistically significant but biologically not relevant increase in lymph node cell count was observed in the mid and highest dose group in comparison to the vehicle control group, however 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 or exceeded in any dose group. Furthermore, stimulation indices of all dose groups were well below the threshold value of 3 for a positive response. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.