Dibutyltin oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 MAY 2021 - 16 FEB 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 July 1992

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Although the murine Local Lymph Node Assay (LLNA) is currently the preferred assay for chemicals under EU REACH regulation, guinea pig assays are equally acceptable under other regulations.
The guinea pig has been found informative for predictive sensitisation tests and is used as animal model since several decades.

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyltin oxide

EC Number:

212-449-1

EC Name:

Dibutyltin oxide

Cas Number:

818-08-6

Molecular formula:

C8H18OSn

IUPAC Name:

dibutylstannanone

Test material form:

solid: particulate/powder

Details on test material:

Chemical name: Dibutyloxostannane
Batch/Lot No.: 1TP0114211
Purity: 97.9 %
Appearance: White powder
Date of manufacture: Sep. 1, 2020
Storage condition: Room temperature (15–25°C), airtight, in a light-resistant container and dehumidification

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Remarks:

ElmSam:HA, SPF

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Samtako Inc., Republic of Korea
- Age at study initiation: 5 weeks
- Weight at study initiation: 297-365 g
- Housing: Polycarbonate cage, W260 × L420 × H180 (mm), one animal per cage, cages and feeders were replaced once weekly.
- Diet (e.g. ad libitum): Pellet feed for experimental animal (Purina experimental diet for guinea pig 38065, Lot No.: P210329WSH, P210529LCS, supplied by the Biopia lnc (LS-ro, Gunpo-si, Gyeonggi-do, Republic of Korea) was provided in feeders ad libitum.
- Water (e.g. ad libitum): Public tap water in Gyeonggi-do was filtered and irradiated by ultraviolet light and provided ad libitum.
- Acclimation period: All animals were quarantined for 5 days (day of animal arrival was considered day 1)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18–22°C
- Humidity (%): 30–70%
- Air changes (per hr): 10–15 times/hr
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle (light on 7 AM to light off 7 PM) (light at 150–300 Lux)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

olive oil

Concentration / amount:

1) water for injection-Freunds Complete Adjuvant (WFI-FCA) emulsion
2) 0.47% test substance
3) 0.47 % test substance-FCA emulsion
each injection 0.1 mL

Day(s)/duration:

on day 0

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: moistened with 0.2 mL water

Concentration / amount:

100 % / 0.2 g

Day(s)/duration:

on day 7 after first induction, dressing removed after 24 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

olive oil

Concentration / amount:

3.13 % / 0.1 mL

Day(s)/duration:

on day 21, dressing was removed after 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 animals in the test group and 10 animals in the (negative) control group

Details on study design:

RANGE FINDING TESTS:
In the preliminary study, 4 male animals were examined in order to select appropriate concentrations for induction and challenge.
Intradermal: The shoulder region of two guinea pig was clipped (JC-4107, JOAS, Republic of Korea) and shaved (SR-292S, JOAS, Republic of Korea) free of hair. Six sites, three each on the left and right sides of the midline of the shaved shoulder region were selected. The left and right sides were injected intradermal with 0.1 mL of the test substance at dose levels of 15, 7.5, 3.75, 1.88, 0.94 and 0.47% 1-mL disposable syringes (26G needle).
Dermal: The left and right flanks of two guinea pig were clipped and closely shaved free of hair. Six areas, on left and right sides of the midline, were selected as application sites. 0.1 g of 100% test substance was applied on a 2×2 cm patch and the patches were placed on the application sites, moistened with 0.1mL of water for injection. 0.1 mL of each test substances was applied at concentrations of 50, 25, 12.5, 6.25 and 3.13% using 1-mL disposable syringes on a 2×2 cm patch and the patches were placed on the application sites. Animals were wrapped with at 7.5 cm adhesive tape (Silkytex, Alcare Co., Ltd., Japan) and the secured with surgical tape (Steri-Drape No. 1040, 3M Co., Ltd., Republic of Korea) for 24 hours occlusive dressing. The occlusive dressing was removed at 24 hours after application. The application sites were gently wiped off with absorbent cotton (DaeHan Medical Supply Co., Ltd., Republic of Korea) moistened with tepid water after patch removal.
The skin reactions were evaluated at 24 and 48 hours after injection and at 24 and 48 hours after patch removal according to the evaluation standard of skin reactions by Magnusson and Kligman.
As a result of the preliminary study, intense redness (score 3) with skin necrosis were observed in both animals at 15, 7.5, 3.75, 1.88 and 0.94% test substances at 24 and 48 hours after intradermal injection. Redness (score 3) was observed at 0.47% test substance.
At 24 and 48 hours after patch removal, redness (score 1) was observed in both animals at 100 and 6.25% test substance. Redness (score 2) was observed in both animals at 50, 25 and 12.5% test substances. The 3.13% test substances did not reveal any evidence of adverse skin reactions in both animals.
In accordance with the result of the preliminary study, the dose level for the first induction was the highest concentration of the test substance at which necrosis was not confirmed at the intradermal injection sites. Therefore, the dose level was selected at 0.47% test substance. Also, the dose level for the second induction will be the highest concentration of the test substance at which mild to moderate skin reactions are confirmed at the topical application sites. Therefore, the dose level was selected at 100% test substance. The challenge dose level will be selected at 3.13% test substance, the highest concentration at which skin reaction is not confirmed at the topical application sites.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: two inductions: intradermal and epicutaneous
- Exposure period: induction on day 0, epicutaneous induction on day 7, for 24 hours
- Test groups:
1) First induction
One day prior to the first induction, the shoulder region of guinea pigs was clipped and shaved free of hair. The first induction day (Day 0), the sensitization site of 2×4 cm area was selected on the left and right by midline. A volume of 0.1 mL of each dose (1-3 from head to tail, see scheme below) to each group with a total six sites, three on the left and right were injected using 1-mL disposable syringes (26G needle):
HEAD
1: WFI-FCA emulsion
2: test substance (0.47%)
3: test substance (0.47%)-FCA emulsion
TAIL
2) Second induction
On Day 6 after the first induction (Day 6), the induction sites were clipped with electric clippers and an electric shaver. On Day 7, an amount of 0.2 g of the 100% test substance and 0.2 mL of moisture water for injection on a 2×4 cm patch (TegadermTM+Pad, No. 3582, 3M Co., Ltd., U.S.A.) was applied to the shoulder region of induction sites of the test substance group.
Patches were wrapped with the expandable adhesive tape and the secured with surgical tape for 48 hours occlusive dressing. Wrappings and patches were removed at 48 hours after application. Then, the application site was wiped clean gently with absorbent cotton moistened with tepid water.
- Control group:
1) First induction
One day prior to the first induction, the shoulder region of guinea pigs was clipped and shaved free of hair. The first induction day (Day 0), the sensitization site of 2×4 cm area was selected on the left and right by midline. A volume of 0.1 mL of each dose (see scheme below) to each group with a total six sites, three on the left and right were injected using 1-mL disposable syringes (26G needle):
HEAD
1: WFI-FCA emulsion
2: olive oil
3: olive oil-FCA emulsion
TAIL
2) Second induction
On Day 6 after the first induction (Day 6), the induction sites were clipped with electric clippers and an electric shaver. On Day 7, a volume of 0.2 mL of olive oil was applied to the induction sites of the control groups.
Patches were wrapped with the expandable adhesive tape and the secured with surgical tape for 48 hours occlusive dressing. Wrappings and patches were removed at 48 hours after application. Then, the application site was wiped clean gently with absorbent cotton moistened with tepid water.
- Concentrations: 0.47 % was used for the first intradermal induction and 100 % was applied dermally for the second induction

B. CHALLENGE EXPOSURE
- No. of exposures: one challenge was performed
- Day(s) of challenge: on day 21 for 24 hours
- Exposure period:
- Test groups: On Day 13 after the second induction (Day 20), the left and right dorsal application sites were trimmed with electric clippers and electric shaver. On Day 21, the shaved areas (2×2 cm) were selected as challenge sites. A volume of 0.1 mL of 3.13% test substance was applied to the left challenge sites of the test substance and control groups. At the right challenge site, 0.1 mL of olive oil was applied. Patches were wrapped loosely with the expandable adhesive tape and then secured with surgical tape for 24 hours occlusive dressing. Wrappings and patches were removed at 24 hours after application. Then, the application sites were wiped clean with absorbent cotton moistened with tepid water.
- Control group: On Day 13 after the second induction (Day 20), the left and right dorsal application sites were trimmed with electric clippers and electric shaver. On Day 21, the shaved areas (2×2 cm) were selected as challenge sites. A volume of 0.1 mL of 3.13% test substance was applied to the left challenge sites of the test substance and control groups. At the right challenge site 0.1 mL of olive oil was applied. Patches were wrapped loosely with the expandable adhesive tape and the secured with surgical tape for 24 hours occlusive dressing. Wrappings and patches were removed at 24 hours after application. Then, the application sites were wiped clean with absorbent cotton moistened with tepid water.
- Evaluation (hr after challenge): Challenge sites were observed visually at 24 and 48 hours after challenge patch removal

Challenge controls:

none

Positive control substance(s):

yes

Results and discussion

Positive control results:

Recent reliability check was performed during the period from Apr 15, 2021 to May 21, 2021. The sensitisation rate after application of the positive control substance was 100%, classified as "V, Extreme", confirming the validity of this test system (results are included below)

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

3.13 % test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No abnormal signs were observed in any animal of each group. All animals of each group exhibited normal body weight gain.

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

3.13 % test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No abnormal signs were observed in any animal of each group. All animals of each group exhibited normal body weight gain.

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

3.13 % test substance

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormal signs were observed in any animal of each group. All animals of each group exhibited normal body weight gain.

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

3.13 % test substance

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormal signs were observed in any animal of each group. All animals of each group exhibited normal body weight gain.

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

10 % hexylcinnamaldehyde

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10 % hexylcinnamaldehyde

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Evaluation of Skin Sensitization

a: test substance group 

Group	Substance for challenge	Animal ID	Score of skin reaction
			24 hours	48 hours
Test substance	Test substance	1101	0	0
		1102	0	0
		1103	0	0
		1104	0	0
		1105	0	0
		1106	0	0
		1107	0	0
		1108	0	0
		1109	0	0
		1110	0	0
		1111	0	0
		1112	0	0
		1113	0	0
		1114	0	0
		1115	0	0
		1116	0	0
		1117	0	0
		1118	0	0
		1119	0	0
		1120	0	0
		Total score	0	0
	control	1101	0	0
		1102	0	0
		1103	0	0
		1104	0	0
		1105	0	0
		1106	0	0
		1107	0	0
		1108	0	0
		1109	0	0
		1110	0	0
		1111	0	0
		1112	0	0
		1113	0	0
		1114	0	0
		1115	0	0
		1116	0	0
		1117	0	0
		1118	0	0
		1119	0	0
		1120	0	0
		Total score	0	0

 

b: control group

Group	Substance for challenge	Animal ID	Score of skin reaction
			24 hours	48 hours
Control group	Test substance	1201	0	0
		1202	0	0
		1203	0	0
		1204	0	0
		1205	0	0
		1206	0	0
		1207	0	0
		1208	0	0
		1209	0	0
		1210	0	0
		Total score	0	0
	control	1201	0	0
		1202	0	0
		1203	0	0
		1204	0	0
		1205	0	0
		1206	0	0
		1207	0	0
		1208	0	0
		1209	0	0
		1210	0	0
		Total score	0	0

Reliability check data (Experimental period: Apr 15, 2021 to May 21, 2021)

Test group	Substance for induction		Substance for challenge (concentration)	Score of skin reaction^a)	Number of animals		Number of animals with positive reactions	Sensitisation rate (%)	Classification of sensitisation grade
	1st (concentration)	2nd (concentration)			24 hours^b)	48 hours^b)
Positive substance	HCA (0.5 %)	HCA (50%)	HCA (10%)	0	0	0	5/5	100	V (Extreme)
				1	1	1
				2	4	4
				3	0	0
				Mean score	1.8	1.8
			Olive oil	0	5	5	0/5	0	I (Negative)
				1	0	0
				2	0	0
				3	0	0
				Mean score	0	0

a): Magnusson & Kligman grading scale

b): Observation after patch removal for challenge

HCA: hexylcinnamaldehyde

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

Based on the result of this study, the test substance, Dibutyltin oxide, did not induce skin sensitization under the conditions of this study.

Executive summary:

This study was conducted in accordance with OECD Guideline 406 and under GLP compliance, to evaluate the skin sensitization potential of the test substance, Dibutyltin oxide, by Maximization test using male Hartley guinea pigs at the age of 5 weeks. Test groups consisted of two groups with a test substance group (20 animals) and control group (10 animals).
In the test substance group, 0.47% test substance was injected intradermally for the first induction. The second induction was conducted with 100% test substance occluded for 48 hours. The challenge was conducted with the 3.13% test substance and olive oil occluded for 24 hours. The skin reaction, such as erythema and swelling, was evaluated at 24 and 48 hours after removal of the challenge patch.
In the control group, the first and second inductions were done with olive oil and the challenges with 3.13% test substance and olive oil. The skin reaction, such as erythema and swelling, was evaluated at 24 and 48 hours after the removal of the challenge patch.
In the test substance and control group, no skin reactions such as erythema and swelling were observed in any animal in test substance and olive oil site at 24 and 48 hours after challenge patch removal.
During the observation period, no abnormal clinical signs or body weight gain was observed in any animal of each group.
Based on the result of this study, the test substance, Dibutyltin oxide, did not induce skin sensitization under the conditions of this study.