Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Principles of method if other than guideline:

The study procedure was extended to include additional measures of lymph node response including lymph node cell count, lymph node weight and ear weight as an indicator of skin irritation to put the data into perspective.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate produced by "Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland Pfalz"

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

EC Number:

915-704-1

Molecular formula:

C36H28ClCrN7O10S2.Na / C34H26Cl2CrN8O8S2.Na / C38H30CrN6O12S2.Na

IUPAC Name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch No. 001-140902
- Purity: 100.0 area-% (sum of all peaks, HPLC)
- Content: 96 g/100 g (100 g/100 g - water)
- Physical stae / color: solid / brown

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was produced on a weight per weight basis shortly before application by stirring with a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspension in MEK (methyl ethyl ketone)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

OlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Postbus 6174, 5960 AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 9 weeks (pretest); 8 weeks (main test)
- Weight at study initiation: 19.2 g – 19.8 g (pretest); 16.9 g – 21.4 g (main test)
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before the first test substance application
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 28 Feb 2017 To:13 Mar 2017

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10 %, 25 % and 50 % test substance in methyl ethyl ketone

No. of animals per dose:

5

Details on study design:

- Compound solubility: The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.
- Randomization: Prior to first application, the animals were distributed between the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of "Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 - 64".
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and / or local inflammation at the application sites were noted for each animal in the raw data.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- Form of application: Epicutaneous application is simulating dermal contact with the compound which possibly occurs under practical use conditions.
- Application volume: 25 µL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site
- 3H thymidine injection: On study day 5 (about 66 to 72 hours after the last application of test substance to the ears), 20 µCi 3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice
- Determination of ear weight: Immediately after the death of each animal (on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia), a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size: 200 µm) into 6 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casyton in a ratio 1:500. The cell count was determined by using a Casy Counter.
- Measurement of 3H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with PBS and precipitated with 5 % trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by the ones of the vehicle control group. Further statistical analyses of 3H-thymidine incorporation, cell count, lymph node weight and ear weight were performed according to WILCOXON-Test.

Results and discussion

Positive control results:

see Tab.2 below

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

10 % test substance in MEK

Remarks on result:

other: SI-increases of the lymph node parameters are not considered to indicate a sensitization reaction but to be caused by the irritation effects

Results 2

Key result

Parameter:

SI

Value:

1.61

Test group / Remarks:

25 % test substance in MEK

Remarks on result:

other: SI-increases of the lymph node parameters are not considered to indicate a sensitization reaction but to be caused by the irritation effects

Results 3

Key result

Parameter:

SI

Value:

2.9

Test group / Remarks:

50 % test substance in MEK

Remarks on result:

other: SI-increases of the lymph node parameters are not considered to indicate a sensitization reaction but to be caused by the irritation effects

Cellular proliferation data / Observations:

When applied as 10 %, 25 % and 50 % preparations in MEK, the test substance did not induce a biologically relevant (no increase above the cut off Stimulation Index of 3) increase of 3H-thymidine incorporation into the cells form the auricular lymph nodes. The increases in lymph node cell counts above a stimulation index of 1.5 at the 25 % and 50 % concentration were not reflected in the lymph node weights as well as the 3H-thymidine incorporation and thus considered as biologically irrelevant.
Statistically significant increases were noted in 3H-thymidine incorporation at 50 % and in lymph node cell counts at all concentrations.
In addition, statistically significant but concentration independent increase in lymph node weights was noted at the 10 % and 25 % concentrations.
The borderline response in 3H-thymidine incorporation and increase in cell counts (SI > 1.5) observed after treatment with the 50 % preparation can be attributed to the SI of two individual animals that concomitantly showed increases in ear weight (SI > 1.25) indicating an irritating response.

Based on the mean values the test-substance concentrations did not cause increases (SI > 1.25) in ear weights, demonstrating the absence of excessive ear skin irritation. However, the mean SI at the 50 % concentration lies at the border of the cut-off value and two individual animals of the 50 % concentration showed SI > 1.25 indicating a relevant irritating response. Statistically significant increases in ear weights were noted at 25 % and 50 % concentrations.

The expected mean body weight gain was generally observed in animals treated with the test substance at 10 % and 25 %. However, in animals treated with the vehicle control or 50 % concentration slightly reduced mean body weight was observed over the study period.

No relevant signs of systemic toxicity were noticed in all animals during general observation. Golden brown discoloration of feces was noted in all animals of the 25 % and 50 % concentration on study day 2 and 5.

Slight brownish discoloration of the ear skin was noted at the 25 % and 50 % concentrations during the observation period. Slight compound residues were observed in all animals at the 50 % concentration on study day 5.

Any other information on results incl. tables

Tab 1: Summary of results

Test Group	Treatment	³H-thymidine incorporation Stimulation Index^1		Cell Count Stimulation Index^1		Lymph Node Weight Stimulation Index^1		Ear Weight Stimulation Index^1
1	vehicle MEK	1.00		1.00		1.00		1.00
2	10% in MEK	1.30		1.30	#	1.21	##	1.02
3	25% in MEK	1.61		1.58	##	1.21	#	1.04	#
4	50% in MEK	2.90	#	1.83	##	1.21		1.23	##

1 test group x / test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Tab 2: Periodic positive control data

Date of performance	Name of test substance	Concentrations	Vehicle	Stimulation Index ^3H-thymidine incorporation^a	Stimulation Index Cell counts	EC 3.0	EC 1.5
Jun 2016	Alpha-Hexylcinnamaldehyde, techn. 85 %	1 %, 5 %, 15 %	MEK (methyl ethyl ketone)	2.60, 7.08, 14.77	1.50, 2.21, 3.01	1.4	1
Jan 2017	Alpha-Hexylcinnamaldehyde, techn. 85 %	1 %, 5 %, 15 %	MEK (methyl ethyl ketone)	1.56, 3.20, 9.16	1.22, 2.0, 3.42	4.5	2.4

^aRatio of test group values to control group values (Stimulation index) greater than 3.0 indicates a positive result

Tab 3: Summary of Pretest

Test group	Treatment	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
1	10 % in MEK	0.81	0.97
2	50 % in MEK	1.17	1.16

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that the test substance does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test substance was assessed by using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 10%, 25% and 50% (w/w) preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application, 20 μCi ³H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring ³H-thymidine incorporation. Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No relevant signs of systemic toxicity were noticed in all animals during general observation. When applied as 10%, 25% and 50% preparations in MEK, the test substance did not induce a biologically relevant (no increase above the cut off Stimulation Index of 3) increase of ³H-thymidine incorporation into the cells from the auricular lymph nodes. The increases in lymph node cell counts above a stimulation index of 1.5 at the 25% and 50% concentration were not reflected in the lymph node weights as well as the ³H-thymidine incorporation and thus considered as biologically irrelevant. Statistically significant increases were noted in ³H-thymidine incorporation at 50% and in lymph node cell counts at all concentrations.

In addition, statistically significant but concentration independent increase in lymph node weights was noted at the 10% and 25% concentrations.

Based on the mean values the test substance concentrations did not cause increases (SI > 1.25) in ear weights, demonstrating the absence of excessive ear skin irritation. However, the mean SI at the 50% concentration lies at the border of the cut-off value and two individual animals of the 50% concentration showed values SI > 1.25 indicating a relevant irritating response. Statistically significant increases in ear weights were noted at 25% and 50% concentrations. Slight brownish discoloration of the ear skin was noted at the 25% and 50% concentrations during the observation period. Slight compound residues were observed in all animals at the 50% concentration on study day 5.

The borderline response in ³H-thymidine incorporation and increase in cell counts (SI > 1.5) observed after treatment with the 50% preparation can be attributed to the SI of two individual animals that concomitantly showed increases in ear weight (SI > 1.25) indicating an irritating response in those animals.

Overall, the SI-increases of the lymph node parameters are not considered to indicate a sensitization reaction but to be caused by the irritation effects.

Thus, it is concluded that the test substance does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.