[No public or meaningful name is available]

Endpoint summary

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:

03-24-2020 to 06-25-2020

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:

OECD Guideline for Testing of Chemicals No. 429, July 22, 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Physical state / color: Liquid / colourless to slightly yellow
Storage conditions: Room temperature, under nitrogen

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd / SPF

Sex:

female

Details on test animals and environmental conditions:

Age on day 0: 8 - 12 weeks
Sex: Female
Supplier: Envigo RMS GmbH
Kreuzelweg 53
NL-5961 NM Horst
Arrival in the test facility: Acclimatization period at least 5 days before the first test
substance application
Identification: The single housed animals were identified by cage cards
Body weight on day 0: 18.6 g – 22.3 g (pretest)
17.2 g – 20.9 g (main test)

Room temperature / relative humidity: The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 45 – 65% for relative humidity. There were no deviations from these ranges, which influenced the results
of the study.
Day / night rhythm: 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Type of cage: Polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany
Enrichment: PLEXX mouse tunnel (red, transparent) and nest building material Nestlets (Ancare, Typ NES 3600) (PLEXX b.v.; AB Elst, Netherlands)

Number of animals per cage: 1 animal
As group housing may increase oral exposure due to grooming of the animals and may interfere with clear observations of each individual animal, animals were single housed for the duration of the test.
Feeding: Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
Drinking water: Tap water ad libitum
Bedding: Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data).

Vehicle:

methyl ethyl ketone

Concentration:

10% and 50% (first pretest)
0.5%, 1.0% and 2.5% (2nd pretest)
0.05%, 0.1% and 0.25% (main study)

No. of animals per dose:

5

Details on study design:

Conduct of the study
The study comprised four test groups. Each group consisted of 5 mice.
Randomization: Prior to first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions of Win Rando Version 3.2.
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 can occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal surface 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), about 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.

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 8 mL 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.

Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using a PC-based tabular calculation software. The mean and individual data were not always rounded, but the significant digits were produced by changing the display format.
Consequently, calculation of mean values by using the individual data presented in the report will in some instances yield minor variations in value.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde, techn. 85%

Statistics:

Mean values and standard deviations of the measured parameters were calculated per test group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.
Statistical analysis of 3H-Thymidine incorporation, cell count, lymph node weight and ear weight were performed by the WILCOXON test.

Positive control results:

Result of last run including a periodic positiv control:
1%, 5%, 15% Alpha-Hexylcinnamaldehyde, techn. 85%
Stimulation Index (3H-Thymidine incorporation): 3.06, 7.70 and 11.13 (ratio of test group values to control group values >=3.0 indicates a positive result.
Stimulation index (cell counts): 1.59, 2.26 and 3.00
EC 3: ca. 1
EC 1.5: ca. 1

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

1

Test group / Remarks:

vehicle MEK

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

2.55

Test group / Remarks:

0.05% in MEK

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

9.1

Test group / Remarks:

0.10% in MEK

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

22.44

Test group / Remarks:

0.25% in MEK

Parameter:

SI

Remarks:

cell counts

Value:

1

Test group / Remarks:

vehicle MEK

Parameter:

SI

Remarks:

cell counts

Value:

1.55

Test group / Remarks:

0.05% in MEK

Parameter:

SI

Remarks:

cell counts

Value:

2.24

Test group / Remarks:

0.10% in MEK

Parameter:

SI

Remarks:

cell counts

Value:

3.53

Test group / Remarks:

0.25% in MEK

Parameter:

EC3

Remarks:

3H-thymidine incorporation

Value:

0.053

Remarks on result:

other: %

Parameter:

other: EC1.5

Remarks:

cell count

Value:

0.048

Remarks on result:

other:

Remarks:

%

Pretest / Irritation Screening

In the pretest two mice each were treated with test-substance concentrations of 10% and 50% (1st pretest) and 0.5%, 1.0% and 2.5% (2nd pretest) on three consecutive days. Overall, no relevant signs of systemic toxicity were observed in the pretests. However, reduced general condition was observed in the animals treated with the 10% and 50% concentrations on study day 2.

After application of the 10% and 50% concentrations, the animals showed severe increases (>25%) in ear weights (compared to historical vehicle values) and ear thickness measurements as indication of excessive ear skin irritation. In addition, well-defined erythema (day 2) and slight scales on the ear skin (day 5) were observed at the 10% concentration. Moderate to severe erythema (day 2) and moderate scales on the ear skin (day 5) were noted in animals treated with the 50% concentration. After treatment with the 1.0% and 2.5% concentrations, the animals showed severe increases (≥25%) in ear thickness measurements as indication of excessive ear skin irritation. Slight scales on the ear skin were observed at the 2.5% concentration on study day 5. The 0.5% concentration did not show relevant signs of local ear skin irritation. Considerably increased lymph node weights were observed in all concentrations without a clear dose-response relationship.

Test Group	Treatment	Animal No.	Lymph Node Weight [mg/Lymph Node Pair]	Mean	S.D.	Stimulation Index^1(SI) per animal	Stimulation Index^1(SI) per test group
	vehicle historical control			5.3	0.3		1.00
1	10% in MEK	1 2	23.8 17.5	20.7	4.5	4.49 3.30	3.90
2	50% in MEK	3 4	22.1 21.8	22.0	0.2	4.17 4.11	4.14
3	0.5% in MEK	5 6	17.7 18.1	17.9	0.3	3.34 3.42	3.38
4	1.0% in MEK	7 8	19.6 20.9	20.3	0.9	3.70 3.94	3.82
5	2.5% in MEK	9 10	19.5 24.7	22.1	3.7	3.68 4.66	4.17

Test Group	Treatment	Animal No.	EarWeight [mg/animal]	Mean	S.D.	Stimulation Index^1(SI) per animal	Stimulation Index^1(SI) per test group
	vehicle historical control			30.9	1.3		1.00
1	10% in MEK	1 2	38.0 40.4	39.2	1.7	1.23 1.31	1.27
2	50% in MEK	3 4	42.9 41.8	42.4	0.8	1.39 1.35	1.37
3	0.5% in MEK	5 6	30.4 31.9	31.2	1.1	0.98 1.03	1.01
4	1.0% in MEK	7 8	34.0 33.5	33.8	0.4	1.10 1.08	1.09
5	2.5% in MEK	9 10	35.9 36.9	36.4	0.7	1.16 1.19	1.18

¹ test group x / vehicle MEK (historical control)

Test Group	Treatment	Animal No.	Ear Thickness [µm]
			d 0			d 2			d 5
			left	right	Mean	left	right	Mean	left	right	Mean
1	10% in MEK	1 2	200 205	200 200	200 203	260 250	260 250	260 250	270 280	270 280	270 280
2	50% in MEK	3 4	200 200	200 200	200 200	270 260	270 260	270 260	340 360	350 360	345 360
3	0.5% in MEK	5 6	200 205	200 205	200 205	225 225	225 225	225 225	240 245	235 240	238 243
4	1.0% in MEK	7 8	200 200	200 200	200 200	225 225	225 225	225 225	250 250	250 255	250 253
5	2.5% in MEK	9 10	205 200	205 200	205 200	245 240	245 245	245 243	270 275	265 270	268 273

Test Group	Treatment	Animal No.	Difference d 0 to d 2 [µm]	Ear swelling d 2 (%)	Difference d 0 to d 5 [µm]	Ear swelling d 5 (%)
1	10% in MEK	1 2	60 48	30 23	70 78	35 38
2	50% in MEK	3 4	70 60	35 30	145 160	73 80
3	0.5% in MEK	5 6	25 20	13 10	38 38	19 18
4	1.0% in MEK	7 8	25 25	13 13	50 53	25 26
5	2.5% in MEK	9 10	40 43	20 21	63 73	30 36

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	10% in MEK	1 2	18.9 18.6	18.8	0.2	19.9 19.2	19.6	0.5	1.0 0.6	0.8	0.3
2	50% in MEK	3 4	21.3 19.3	20.3	1.4	20.5 19.3	19.9	0.8	-0.8 0.0	-0.4	0.6
3	0.5% in MEK	5 6	20.1 18.7	19.4	1.0	19.9 19.8	19.9	0.1	-0.2 1.1	0.4	0.9
4	1.0% in MEK	7 8	20.0 22.1	21.1	1.5	19.8 21.9	20.9	1.5	-0.2 -0.2	-0.2	0.0
5	2.5% in MEK	9 10	22.3 21.0	21.7	0.9	23.3 21.9	22.6	1.0	1.0 0.9	0.9	0.1

Nature and duration of clinical findings of individual animals:

Test Group	1
Treatment	10% in MEK
Animal Numbers	1	2
reduced general condition	d2	d2

 

Test Group	2
Treatment	50% in MEK
Animal Numbers	3	4
reduced general condition	d2	d2

 

Test Group	3
Treatment	0.5% in MEK
Animal Numbers	5	6
	-	-

 

Test Group	4
Treatment	1.0% in MEK
Animal Numbers	7	8
	-	-

 

Test Group	5
Treatment	2.5% in MEK
Animal Numbers	9	10
	-	-

 

Nature and duration of local findings of individual animals

Test Group	1
Treatment	10% in MEK
Animal Numbers	1	2
well-defined erythema	d2	d2
scales on the ear skin, slight	d5	d5

 

Test Group	2
Treatment	50% in MEK
Animal Numbers	3	4
moderate to severe erythema	d2	d2
scales on the ear skin, moderate	d5	d5

 

Test Group	3
Treatment	0.5% in MEK
Animal Numbers	5	6
	-	-

 

Test Group	4
Treatment	1.0% in MEK
Animal Numbers	7	8
	-	-

 

Test Group	5
Treatment	2.5% in MEK
Animal Numbers	9	10
scales on the ear skin, slight	d5	d5

 

Main test

3H-thymidine incorporation, cell count and lymph node weight

When applied as 0.10% and 0.25% concentrations in MEK, the test substance induced biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and concentration-dependent increases of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.05% concentration failed to reach the cutoff value. The increases of 3H-thymidine incorporation were statistically significant at all concentrations.

Concomitantly, all concentrations induced biologically relevant and statistically significant responses (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The increase of the 0.05% concentration lies at the border of of biological relevance. In addition, statistically significant increases in lymph node weights were noted at all concentrations.

 

Test Group	Treatment	³H-thymidine incorporation [DPM/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	120.9	45.4	1.00
2	0.05% in MEK	308.1	59.2	2.55	##
3	0.10% in MEK	1,099.6	340.3	9.10	##
4	0.25% in MEK	2,712.2	445.9	22.44	##

 

 

Test Group	Treatment	Cell Counts [Counts/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	9,952,800	1,624,907	1.00
2	0.05% in MEK	15,420,800	1,836,718	1.55	##
3	0.10% in MEK	22,309,600	4,161,851	2.24	##
4	0.25% in MEK	35,161,600	7,514,526	3.53	##

 

 

Test Group	Treatment	Lymph Node Weight [mg/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	5.2	0.9	1.00
2	0.05% in MEK	7.0	1.0	1.34	##
3	0.10% in MEK	9.3	1.3	1.77	##
4	0.25% in MEK	14.3	0.8	2.73	##

¹vs. mean of test group 1 (vehicle control)

## = statistically significant for the value p ≤ 0.01

Ear weights

The test-substance concentrations did not cause any increases in ear weights (no increase to stimulation index (SI) of ≥ 1.25), demonstrating the absence of relevant ear skin irritation.

Test Group	Treatment	Ear Weight [mg/animal]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	31.1	1.8	1.00
2	0.05% in MEK	30.0	1.1	0.97
3	0.10% in MEK	30.6	0.9	0.98
4	0.25% in MEK	31.4	1.3	1.01

¹ vs. mean of test group 1 (vehicle control)

## = statistically significant for the value p ≤ 0.01

Body weights

The expected mean body weigh gain was generally observed in all test groups during the study period.

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	17.7			19.1			1.4
		2	17.2			17.8			0.6
1	vehicle MEK	3	19.5	18.8	1.4	19.5	19.1	1.1	0.0	0.2	0.8
		4	19.2			18.4			-0.8
		5	20.6			20.6			0.0
		6	20.0			21.2			1.2
		7	19.6			19.4			-0.2
2	0.05% in MEK	8	19.4	19.3	1.0	20.3	19.7	1.3	0.9	0.4	0.6
		9	17.5			17.7			0.2
		10	19.9			19.8			-0.1
		11	19.8			20.9			1.1
		12	18.7			19.4			0.7
3	0.10% in MEK	13	19.2	19.3	0.5	20.1	20.2	0.9	0.9	0.9	0.4
		14	19.1			19.4			0.3
		15	19.9			21.4			1.5
		16	19.2			21.0			1.8
		17	20.4			22.1			1.7
4	0.25% in MEK	18	18.1	19.5	1.1	19.0	20.7	1.1	0.9	1.1	0.7
		19	20.9			21.0			0.1
		20	19.0			20.2			1.2

Clinical signs

No signs of systemic toxicity were noticed in all animals during general observation.

Local findings

No local findings were observed during the observation period.

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Basonat HA 3000 exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of Basonat HA 3000 was assessed using the radioactive Local Lymph Node Assay. 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 0.05%, 0.10% and 0.25% (w/w) preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, about 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 measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count and weight 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 ear skin irritation. No signs of systemic toxicity or irritation were noticed in all animals during general observation.

When applied as 0.10% and 0.25% concentrations in MEK, the test substance induced biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and concentration-dependent increases of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.05% concentration failed to reach the cutoff value. The increases of 3H-thymidine incorporation were statistically significant at all concentrations. Concomitantly, all concentrations induced biologically relevant and statistically significant responses (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The increase of the 0.05% concentration lies at the border of of biological relevance. In addition, statistically significant increases in lymph node weights were noted at all concentrations.

The test-substance concentrations did not cause any increases in ear weights (no increase to stimulation index (SI) of ≥ 1.25), demonstrating the absence of relevant ear skin irritation.

Thus, it is concluded that Basonat HA 3000 exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was around 0.05%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 0.05% and 0.10% concentrations to be 0.053%. The EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semi-logarithmical regression from the results of the 0.05% and 0.10%

concentrations to be 0.048%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of Basonat HA 3000 was assessed using the radioactive Local Lymph Node Assay according to OECD TG 429. When applied as 0.10% and 0.25% concentrations in MEK, the test substance induced biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and concentration-dependent increases of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.05% concentration failed to reach the cutoff value. The increases of 3H-thymidine incorporation were statistically significant at all concentrations. Concomitantly, all concentrations induced biologically relevant and statistically significant responses (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The increase of the 0.05% concentration lies at the border of of biological relevance. In addition, statistically significant increases in lymph node weights were noted at all concentrations.

The test-substance concentrations did not cause any increases in ear weights (no increase to stimulation index (SI) of ≥ 1.25), demonstrating the absence of relevant ear skin irritation.

Thus, it is concluded that Basonat HA 3000 exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was around 0.05%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 0.05% and 0.10% concentrations to be 0.053%. The EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semi-logarithmical regression from the results of the 0.05% and 0.10% concentrations to be 0.048%.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a state-of-the-art LLNA according to OECD TG 429, the substance showed EC3 values <2%. According to Regulation (EC) No 1272/2008, Annex I, classification is required as skin sensitising Cat.1A; H317: May cause an allergic skin reaction.