2,4,8,10-tetraoxa-3λ6,9λ6-dithiaspiro[5.5]undecane 3,3,9,9-tetraoxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Buehler Test Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 August 2022 to 23 November 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted on 17 July 1992 (Corrected on 30 June 2022)

Deviations:

yes

Remarks:

One deviation: minimum temperature maintained was between 20+3°C except from 11/08/2022 to 15/08/2022, where in minimum temperature was 19.6°C to 19.9°C. This deviation does not have any impact on the outcome of the study as all animals were normal.

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The LLNA method was not available yet by the time the study was conducted.
The study was performed in an AAALAC accredited facility:
a.In accordance with the recommendation of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for laboratory animal facility published in the Gazette of India, 2018.
b.In accordance with the protocol approved by Institutional Animal Ethics Committee (IAEC) (Protocol No.: BIO-IAEC-4452 and Approval Date: 04/06/2022).

Test material

Test material information

Constituent 1

Reference substance name:

2,4,8,10-tetraoxa-3λ⁶,9λ⁶-dithiaspiro[5.5]undecane-3,3,9,9-tetrone

EC Number:

859-869-7

Cas Number:

201419-80-9

Molecular formula:

C5H8O8S2

IUPAC Name:

2,4,8,10-tetraoxa-3λ⁶,9λ⁶-dithiaspiro[5.5]undecane-3,3,9,9-tetrone

Test material form:

solid

Specific details on test material used for the study:

Name of Test Item : 2,4,8,10-Tetraoxa-3,9-dithiaspiro [5.5]unde cane 3,3,9,9-tetraoxide
Chemical Name (IUPAC) : 2,4,8,10-Tetraoxa-3,9-dithiaspiro [5.5]unde cane 3,3,9,9-tetraoxide
CAS No. : 201419-80-9
Physical appearance (with colour): White solid
Purity (as per Certificate of Analysis): 99.9%
Lot No. : S022012901
Date of Manufacture : 2022.1.29
Date of Expiry : 2023.3.29
Storage Conditions : Cool and dry (+2 to +8ºC)

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

Cavia porcellus

Sex:

female

Details on test animals and environmental conditions:

No. of Groups: Pre-Study : 1
Main Study: 2
No. of Animals / Group and Sex:
Pre-Study:
G1 - 2 Females
Main Study:
G2 - 10 Females - Vehicle Control
G3 - 20 Females - Test Item
(Females used were nulliparous and non-pregnant)
Age at Receipt: 8 weeks
Body Weight Range at Receipt: 321.02 g to 340.10 g
Animal Identification: Acclimatization period: Cage cards
Treatment period: Animal identification was done by writing last two digits of animal number on ear of guinea pig using black marker pen and cage cards

Husbandry
a. Environmental Conditions:
Animals were housed under standard laboratory conditions, in an environmentally monitored, air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour) room temperature 19.6°C to 22.9ºC, relative humidity 47% to 66%, with 12 hours light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
b. Housing:
Single animal was housed in a standard polycarbonate cage (size: L 430 x B 280 x H 210 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized corn cob was provided as bedding material.
c. Feed:
Altromin diet for guinea pigs (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
d. Water:
Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through Reverse osmosis unit was provided in plastic water bottle with stainless steel sipper tubes.

Acclimatization:
Healthy young adult animals were acclimatized for five days and twelve days to experimental room conditions prior to treatment for pre-study and main study respectively and were observed for clinical signs once daily. Veterinary examination of all the animals was performed on the day of receipt.

Grouping
The animals for the main study were weighed and arranged in ascending order of their body weights. These body weight stratified Guinea pigs were distributed to both the experimental groups using microsoft excel spreadsheet such that body weight variation of animals selected for the experiment did not exceed ±20% (-3.05% to +2.97%) of the mean body weight. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups.

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

other: 80% ethanol/water

Concentration / amount:

The animals did not reveal any skin reactions after application of 100 mg of test item moistened with 0.1 mL of 80% ethanol/water at 24 hours and 48 hours after patch removal in the pre-study. Hence, 100 mg of test item moistened with 0.1 mL of 80% ethanol/water was selected for induction.
Dose concentrations prepared for pre-study: 25%, 50%, 75%, 100% w/v Test Item in vehicle

Day(s)/duration:

induction day 1, 8 and 15

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Concentration / amount:

The animals did not reveal any skin reactions after application of 100 mg of test item moistened with 0.1 mL of 80% ethanol/water at 24 hours and 48 hours after patch removal in the pre-study. Hence, 100 mg test item moistened with 0.1 mL of acetone for challenge phase of the main study.

Day(s)/duration:

challenge on day 29

No. of animals per dose:

Pre-Study:
G1 - 2 Females
Main Study:
G2 - 10 Females - Vehicle Control
G3 - 20 Females - Test Item

Details on study design:

Pre-Study:
The objective of the pre-study was to determine the appropriate concentrations of test item that was employed in the main study.
No skin reactions were observed at any of the test doses, hence highest dose of the test item was selected for induction and challenge phase of the main study.

Preparation of Animals:
Approximately 24 hours before application, for induction and challenge phase; the fur from the left flank region and right flank region of the Guinea pigs was closely clipped using an electric hair clipper respectively, exposing an area of approximately 80 sq. cm. Care was taken to avoid abrasion to the skin.

Application of Test Item:
The test item was applied on to the pre-clipped area of the skin. Approximately 4×6 cm^2 of the cotton gauze was covered on the test item applied area of the skin and held in place with non-irritating adhesive tape. The application site was wrapped with crepe bandage for the duration of the exposure period in order to avoid the access by animal to the patch and ingestion or inhalation of the test item. The test patch was held in its position for 6 hours, after which the test patches were removed and the area was cleaned with normal saline swabs and dried with cotton.

Treatment:
Pre-Study:
The weighed test item was moistened with 0.1 mL of 80% ethanol/water and applied topically to the pre-clipped area of the pre-study animals at four different sites.
Main Study:
I) Induction
In the induction phase, 100 mg of test item moistened with 0.1 mL of 80% ethanol/water was applied topically on day 1, 8 and 15 on the same area of left flank region for all the animals of G3 group.
Approximately 24 hours before each treatment, the fur from the application site (left flank) was closely clipped as per the section 6.12 in both vehicle control and treatment group animals. For G3 group animals, 100 mg of test item moistened with 0.1 mL of 80% ethanol/water and applied on to the pre-clipped area of the skin and covered by approximately 4 cm × 6 cm cotton gauze patch. Similarly for G2 group animals, 0.1 mL of the vehicle (80% ethanol/water) was applied on to the pre-clipped area of the skin and covered by approximately 4 cm × 6 cm cotton gauze patch.
II) Challenge
Approximately 24 hours before the application of the test item, the untreated right flank of the Guinea pigs was clipped closely. Both vehicle control and treatment group (G2 and G3) animals were challenged on day 29. 100 mg of test item selected from the pre-study was moistened with 0.1 mL of acetone and applied to the posterior part of the untreated right flank and 0.1 mL of vehicle (acetone) was applied to the anterior part of the same flank. The test patches were covered by approximately 4 cm × 6 cm cotton gauze and held in place with non-irritating adhesive tape and crepe bandage. The test patch was kept in contact with the skin for a period of 6 hours, after which the test patch was removed and the area was cleaned with normal saline swab and dried.
The application sites were examined approximately at 24 hours (i.e., 30 hours after challenge application) and 48 hours (54 hours after the challenge application) after patch removal for skin reaction and scored according to Magnusson and Kligman grading scale.

Observations:
1) Clinical Signs of Toxicity and Mortality: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity during the experimental period.
2) Skin Reactions Scoring
a) Pre-Study: The skin reactions were observed approximately at 24 and 48 hours post removal of the test patch. The skin reactions were observed and recorded according to Draize method (1959).
b) Main Study
i) Induction
The skin reactions were scored approximately at 1 and 24 hours post removal of the test patch after induction (topical application). The skin reactions were observed and recorded according to Draize method (1959).
ii) Challenge
The skin reactions were scored approximately at 24 and 48 hours post removal of the test patch after challenge application. The skin reactions were observed and recorded according to Magnusson and Kligman grading scale.
3) Body Weight
Individual animal body weight was recorded at receipt, on day 1 (before start of the treatment) and at termination for the pre-study and main study animals.

Challenge controls:

Vehicle Control- 10 Females
Treatment- 20 Females
Challenge on day 29
Dose: Anterior Part of Right flank- 0.1 mL of acetone
Posterior Part of Right flank- 100 mg of test item moistened with 0.1 mL of acetone

Positive control substance(s):

no

Remarks:

The positive control was not included in this study as the reliability of the skin sensitisation was tested using 2-Mercaptobenzothiazole under separate study, which was conducted during 31 January 2022 to 12 March 2022.

Results and discussion

Positive control results:

The positive control was not included in this study as the reliability of the skin sensitisation was tested using 2-Mercaptobenzothiazole under separate study. Based on the results of the separate study, it is concluded that "2-Mercaptobenzothiazole - 97%" showed a mean skin sensitisation rate of 60% in topical challenge phase. Hence, the test item is classified as a "Sensitiser (Sub-category 1B)" according to the GHS of classification and labelling of chemicals.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 (0.1 mL of acetone)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were observed

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 (0.1 mL of acetone)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were observed

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% w/v Test Item in vehicle (100 mg of test item moistened with 0.1 mL of acetone)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No skin reactions were observed

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% w/v Test Item in vehicle (100 mg of test item moistened with 0.1 mL of acetone)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No skin reactions were observed

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50 mg test item moistened with 0.1 mL of acetone

No. with + reactions:

13

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Remarks:

At challenge phase, the skin reactions like discrete or patchy erythema to no erythema was observed at posterior part of right flank of 2-MBT application site in 13/20 animals (i.e. 65%) approximately at 24 hours and 11/20 animals (i.e. 55%) approximately at 48 hours post patch removal, the mean sensitization rate was 60%. Based on the above results of the experiment and under experimental conditions employed, it is concluded that “2-Mercaptobenzothiazole - 97%” showed a mean skin sensitisation rate of 60% in topical challenge phase. Hence, the test item is classified as a “Sensitiser (Sub-category 1B)” according to the GHS.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50 mg test item moistened with 0.1 mL of acetone

No. with + reactions:

11

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Remarks:

At challenge phase, the skin reactions like discrete or patchy erythema to no erythema was observed at posterior part of right flank of 2-MBT application site in 13/20 animals (i.e. 65%) approximately at 24 hours and 11/20 animals (i.e. 55%) approximately at 48 hours post patch removal, the mean sensitization rate was 60%. Based on the above results of the experiment and under experimental conditions employed, it is concluded that “2-Mercaptobenzothiazole - 97%” showed a mean skin sensitisation rate of 60% in topical challenge phase. Hence, the test item is classified as a “Sensitiser (Sub-category 1B)” according to the GHS.

Any other information on results incl. tables

Clinical Signs of Toxicity and Mortality:

No clinical signs of toxicity and mortality were observed in both the pre study and main study animals.

 

Skin Reactions Scoring:

No erythema and oedema were observed in G1 group animals at any of the doses tested approximately at 24 and 48 hours after patch removal in pre study.
No erythema and oedema were observed in both the vehicle control (G2) and treatment group (G3) animals in all the induction phases approximately at 1 and 24 hours observation after patch removal in main study.
No skin reactions were observed in both the vehicle control (G2) and treatment group (G3) animals in challenge phase approximately at 24 hours and 48 hours observations after patch removal in main study.

Refer Tables 3 and 4

• Tables 3 SKIN REACTIONS SCORING RECORD DURING PRE-STUDY

Group, Sex & Treatment	Animal No.	Site	Dose (mg)	24 hours*		48 hours*
				Ery	Ede	Ery	Ede
G1,  Female & (2,4,8,10-Tetraoxa-3,9-dithiaspiro [5.5]unde cane 3,3,9,9-tetraoxide)	Gf2175	ALF	25.0	0	0	0	0
		PLF	50.0	0	0	0	0
		ARF	75.0	0	0	0	0
		PRF	100.1	0	0	0	0
	Gf2176	ALF	25.2	0	0	0	0
		PLF	50.0	0	0	0	0
		ARF	75.0	0	0	0	0
		PRF	100.1	0	0	0	0

Ery: Erythema; Ede: Oedema; ALF: Anterior Left Flank; PLF: Posterior Left Flank; ARF: Anterior Right Flank;

PRF: Posterior Right Flank; 0: No erythema/No oedema

*: Observation time points after removal of the test patch

 

• Tables 4 SKIN REACTIONS SCORING RECORD DURING PRE-STUDY

Group, Sex & Treatment	Animal No.	Induction Phase												Challenge Phase Day 29
		Induction I - Day 1				Induction II - Day 8				Induction III - Day 15				Skin Reaction Score (Right Flank)				Sensitisation Rate (%)
		Skin Reaction Score (Left Flank)				Skin Reaction Score (Left Flank)				Skin Reaction Score (Left Flank)
		1 hr*		24 hrs*		1 hr*		24 hrs*		1 hr*		24 hrs*		24 hrs*		48 hrs*
		Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Post	Ant	Post	Ant
G2, Female & Vehicle Control	Gf2177	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2178	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2179	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2180	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2181	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2182	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2183	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2184	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2185	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Gf2186	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Ery: Erythema; Ede: Oedema; hr(s): Hour(s); 0: No Erythema/Oedema; Post: Posterior part (test item); Ant: Anterior part (vehicle control); 0: No visible change for challenge scoring

*: Observation time points after removal of the test patch.

 

Body Weight:

No treatment related change in body weight and percent change in body weight with respect to day 1 in any of the animals. All the animals showed physiologically normal increase in body weights in pre study and main study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions employed and based on the results of the experiment, it is concluded that the test item (2,4,8,10-Tetraoxa-3,9-dithiaspiro [5.5]unde cane 3,3,9,9-tetraoxide) did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the test item does not meet classification criteria according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.

Executive summary:

The study was conducted to evaluate the skin Sensitisation potential of the test item (2,4,8,10-Tetraoxa-3,9-dithiaspiro [5.5]unde cane 3,3,9,9-tetraoxide) in Guinea Pigs by Buehler Test Method.

The study was conducted in two phases viz., pre-study and the main study. For pre-study, approximately 24 hours before treatment, fur from the right and left flank region was clipped closely using an electric hair clipper exposing an area of approximately 80 sq. cm. The dose of 25 mg and 50 mg of test item (moistened with 0.1 mL of 80% ethanol/water) were applied topically to the anterior left flank and posterior left flank respectively, whereas 75 mg and 100 mg of test item (moistened with 0.1 mL of 80% ethanol/water) were applied topically to the anterior right flank and posterior right flank respectively to each animal. No skin reactions were observed in any of the test dose approximately at 24 and 48 hours observation period after patch removal.

In pre-study (G1), no skin reactions were observed at the highest dose i.e. 100 mg of test item. Hence, 100 mg of test item was selected for induction and challenge phase of main study.

The main study comprised of two groups, G2 (Vehicle control) and G3 (treatment). The main study included three inductions on day 1, 8 and 15 and challenge on day 29. Approximately 24 hours prior to initiation of the treatment, fur from the left and right flank was clipped closely using an electric hair clipper exposing an area of approximately 80 sq. cm. for induction and challenge phase of the experiment respectively.

On induction day 1, 8 and 15, 0.1 mL of 80% ethanol/water and 100 mg of test item moistened with 0.1 mL of 80% ethanol/water were applied topically on left flank region to G2 and G3 group animals respectively. On challenge day 29, 0.1 mL of acetone was topically applied to the anterior part of right flank, whereas 100 mg of test item moistened with 0.1 mL of acetone was topically applied to the posterior part of the right flank of all the animals. The test patches were covered by approximately 4 cm × 6 cm cotton gauze and held in place with non-irritating adhesive tape and crepe bandage. After 6 hours of contact period, the test patches were removed and the application sites were cleaned with normal saline swabs and dried. During induction phase, the test item application sites were observed for skin reactions approximately at 1 and 24 hours post removal of the test patches according to Draize method (1959) and during challenge phase the skin reactions were observed approximately at 24 and 48 hours post removal of the test patch according to Magnusson and Kligman grading scale.

All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity during the experimental period. Individual animal body weight was recorded at receipt and on day 1 (before start of the treatment) and at termination for the pre-study and main study animals.

In induction phase, no skin reactions were observed in vehicle control and treatment group animals approximately at 1 and 24 hours of observation period of post patch removal.

In challenge phase, the animals did not reveal any skin reactions at anterior and posterior part of right flank of vehicle control and treatment group animals approximately at 24 and 48 hours of post patch removal.

No clinical signs of toxicity and mortality were observed till termination. All the treated animals revealed physiologically normal increase in body weights during the observation period.