Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1)

Endpoint summary

Description of key information

In an LLNA according to OECD Guideline 429, the test substance did not show a skin sensitising potential (BASF Colors&Effects, 2017).

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:

2016-08-10 to 2017-08-11

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:

2010-07-22

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:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 002-141407

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / orange

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V.,Inc., 5960 AD Horst, The Netherlands
- Age at study initiation: 8 - 10 weeks (pretest), 8 weeks (main test)
- Weight at study initiation: 18.1 - 21.4 g (pretest), 16.4 - 22.0 g (main test)
- Housing: The animals were single housed in fully air-conditioned rooms.
- Diet: ad libitum, Kliba mouse/rat maintenance diet “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland
- Water: ad libitum, drinking water
- Acclimation period: at least 5 days before the first test-substance application.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70

- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

pretest: 10, 25, and 50 %
main test: 2.5, 5, and 10 %

No. of animals per dose:

pretest: 2
main test: 5

Details on study design:

PRE-SCREEN TESTS:
- Irritation: no relevant signs of local irritation were observed at 10 %
- Systemic toxicity: mortality at 25 and 50 %

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-thymidine incorporation, cell count and lymph node weight
- Criteria used to consider a positive response:
In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal surface of both ears was determined by measuring 3H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are, lymph node cell count and, to a certain extent, lymph node weight. Because irritation by the test substance may also induce lymph node responses, the weights of ear punches taken from the area of test-substance application were determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance.
A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.
If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.

TREATMENT PREPARATION AND ADMINISTRATION:
The test-substance preparations were produced on a weight per weight (w/w) 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.

Conduct of the study:
The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice.
Randomization:
Prior to first application, the animals were distributed to 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 is possible to occur 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 five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 µCi of 3H-thymidine in 250 µL of sterile saline.

Terminal procedures:
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
Determination of ear weight: Immediately after the death of each animal 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 of phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined 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 β-scintillation counter.

Positive control substance(s):

other: A concurrent positive control (reliability check) with a known sensitizer was not included in this study.

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 those of the vehicle control group.
3H-thymidine incorporation, cell count, lymph node weight and ear weight were statistically analyzed by WILCOXON - Test.

Key result

Parameter:

SI

Value:

< 3

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
there were no biologically relevant or statistically significant increases in lymph node cell counts (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) at all concentrations.

CLINICAL OBSERVATIONS:
No relevant signs of systemic toxicity were noticed in all animals during general observation. Reddish discolored feces were observed in all test-substance treated animals on study day 2.

BODY WEIGHTS
The expected mean body weight gain was generally observed during the study for animals treated with the vehicle control or with the test substance at 2.5 % and 5 %. However, at the 10 % concentration a mean body weight loss was observed.

Table 1: 3H-thymidine incorporation, cell count and lymph node weight: test group mean values, standard deviations and stimulation indices

Test Group	Treatment	3H-thymidine incorporation [DPM/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	Vehicle DMF	966.5	587.8	1.00
2	2.5 % in DMF	1231.2	559.3	1.27	N.S.
3	5 % in DMF	891.1	303.3	0.92	N.S.
4	10 % in DMF	1186.6	157.5	1.23	N.S.

 

Test Group	Treatment	Cell Counts [Counts/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	Vehicle DMF	14180400	1504473	1.00
2	2.5 % in DMF	14415600	1491616	1.02	N.S.
3	5 % in DMF	12084000	1048370	0.85	N.S.
4	10 % in DMF	11155200	1427984	0.79	N.S.

 

Test Group	Treatment	Lymph Node Weight [mg/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	Vehicle DMF	5.7	0.6	1.00
2	2.5 % in DMF	5.7	0.4	0.99	N.S.
3	5 % in DMF	5.3	0.3	0.93	N.S.
4	10 % in DMF	5.3	0.4	0.93	N.S.

 

 Table 2: Ear weight: test group mean values, standard deviations and stimulation indices 

Test Group	Treatment	Ear Weight [mg/animal]
		Mean	S.D.	Stimulation Index^1
1	Vehicle DMF	30.7	1.4	1.00
2	2.5 % in DMF	32.5	2.7	1.06	N.S.
3	5 % in DMF	31.6	1.9	1.03	N.S.
4	10 % in DMF	31.0	1.9	1.01	N.S.

 

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

#          statistically significant for the value p ≤ 0.05

##        statistically significant for the value p ≤ 0.01

N.S.     Not Significant

 

 Table 3: 3H-thymidine incorporation, cell count, lymph node weight and ear weight: individual values 

Test Group	Treatment	Animal No.	3H-thymidine incorporation [DPM/ Lymph Node Pair]	Cell Counts/ Lymph Node Pair	Lymph Node Weight [mg/ Lymph Node Pair]	Ear Weight [mg/animal]
1	Vehicle DMF	1 2 3 4 5	741.6 510.3 1996.1 827.7 756.7	15816000 12228000 15318000 13086000 14454000	6.5 5.4 5.8 4.9 5.9	30.3 29.1 31.1 30.1 32.9
2	2.5% in DMF	21 22 23 24 25	1903.5 720.6 622.2 1638.7 1271.0	13968000 15222000 12150000 16110000 14628000	6.0 5.3 5.3 6.1 5.6	36.5 29.8 30.7 31.7 33.6
3	5% in DMF	26 27 28 29 30	572.9 803.0 1384.1 769.8 925.9	10584000 12096000 12900000 11628000 13212000	5.2 4.8 5.4 5.4 5.7	32.6 34.4 29.7 30.2 31.1
4	10% in DMF	31 32 33 34 35	1209.9 911.5 1276.2 1297.5 1238.1	10020000 10200000 11958000 10302000 13296000	5.2 5.4 5.5 4.6 5.8	32.9 28.7 30.8 29.5 32.9

 

 Table 4: Body weight data 

Test Group	Treatment	Animal No.	Body Weight d0 [g]			Body Weight d5 [g]			Body Weight Change d5-d0 [g]
			Individual	Mean	S.D.	Individual	Mean	S.D.	Individual	Mean	S.D.
1	VehicleDMF	1 2 3 4 5	17.7 19.9 16.4 16.9 18.8	17.9	1.4	19.7 19.5 17.4 17.8 20.3	18.9	1.3	2.0 -0.4 1.0 0.9 1.5	1.0	0.9
2	2.5%inDMF	21 22 23 24 25	18.7 20.3 18.7 18.5 20.4	19.3	0.9	19.3 21.3 19.3 20.2 21.3	20.3	1.0	0.6 1.0 0.6 1.7 0.9	1.0	0.5
3	5% in DMF	26 27 28 29 30	19.9 17.2 19.3 19.0 21.5	19.4	1.6	20.9 18.1 19.7 19.9 21.3	20.0	1.2	1.0 0.9 0.4 0.9 -0.2	0.6	0.5
4	10% in DMF	31 32 33 34 35	18.8 18.1 22.0 17.4 18.5	19.0	1.8	18.8 17.7 20.6 17.0 18.2	18.5	1.4	0.0 -0.4 -1.4 -0.4 -0.3	-0.5	0.5

 

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine Local Lymph Node Assay according to OECD Guideline 429 (BASF Colors&Effects, 2017). The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 2.5 %, 5 % and 10 % w/w preparations of the test substance in dimethylformamide (DMF) or with the vehicle alone. The high concentration was selected based on the presence of systemic toxicity in a pretest using 50 % and 25 % test substance preparations in dimethylformamide (DMF).

The study used 3 test groups and 1 control group. Each test animal was treated with 25μL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25μL per ear of the vehicle alone.

Three days after the last application the mice were injected into the tail vein with 20μCi of 3H- thymidine in 250μL of sterile saline. 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 (indicator of cell proliferation). 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 in order to obtain an indication of possible skin irritation.

The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

 

Test Group	Treatment	³H-thymidine incorporation Stimulation Index*		Cell Count Stimulation Index*		Lymph Node Weight Stimulation Index*		Ear Weight Stimulation Index*
1	vehicle DMF	1.00		1.00		1.00		1.00
2	2.5 % in DMF	1.27	N.S.	1.02	N.S.	0.99	N.S.	1.06	N.S.
3	5 % in DMF	0.92	N.S.	0.85	N.S.	0.93	N.S.	1.03	N.S.
4	10 % in DMF	1.23	N.S.	0.79	N.S.	0.93	N.S.	1.01	N.S.

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 ) ; N.S. = Not Significant

 

No relevant signs of systemic toxicity were noticed in all animals during general observation. However, the mean body weight of test group 4 (10 % concentration) decreased over the study period. When applied as 2.5 %, 5 % and 10 % preparations in DMF, the test substance did not induce abiologically relevant (no increase above the cut off Stimulation Index of 3) or statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, there were no biologically relevant or statistically significant increases in lymph node cell counts (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) at all concentrations. There were no increases in lymph node weights at all concentrations as well. The ear weights of all test-substance treated animals were not relevantly elevated as compared to the vehicle control group demonstrating the absence of ear skin irritation. Red discoloration of the ear skin of the test-substance treated animals was noted from study day 1 up to study day 5. In addition, encrusted compound residues (slight to severe) around the application site were observed in all test-substance treated animals on study day 5. The animals of the vehicle control group showed slight scaling on study day 5.

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.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

No skin sensitising properties were observed. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.