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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 August 2022 to 22 October 2022

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 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted on 09 October 2017 (Corrected on 14 June 2021)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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)

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Remarks:

Oryctolagus cuniculus

Details on test animals or tissues and environmental conditions:

No. of Animals / Test : Initial Test : 1 Female
Confirmatory Test : 2 Females
(Females used were nulliparous and non-pregnant)
Age at Receipt : 4 to 5 months
Body Weight at Receipt : 2.21198 kg to 2.25014 kg
Animal Identification : Acclimatization: Cage cards
Treatment Period: Cage cards and last 4 digits of the animal number was written on the ear of each rabbit using black permanent marker pen.
Animal No’s: Nc3679 to Nc3681
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 19.7°C to 22.7°C, and relative humidity 48% to 64%, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity was recorded once daily.
b. Housing : The animals were housed individually in stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches) having facilities for holding pelleted feed and drinking water.
c. Feed : Altromin Maintenance diet for rabbits – Rich in crude fibre 2123 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rabbits 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 bottles with stainless steel sipper tubes.

Acclimatization:
Healthy young adult animals were acclimatized for a period of six days and thirteen days for initial and confirmatory test respectively to the experimental room conditions prior to treatment and were observed for clinical signs once daily. Veterinary examination of all the animals was performed on the day of receipt.

Preparation of Animals
Both the eyes of each experimental animal selected for testing were examined within 24 hours before initiating the treatment using ophthalmoscope. Only those animals with absence of signs of eye irritation, ocular defects or pre-existing corneal injury were used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent negative control

Amount / concentration applied:

volume of 0.1 mL (Initial test: 97.2 mg and Confirmatory test: 95.7 mg and 98.2 mg) of test item was instilled

Duration of treatment / exposure:

1 hour treatment

Observation period (in vivo):

72 hours after test item instillation

Number of animals or in vitro replicates:

Initial Test : 1 Female
Confirmatory Test : 2 Females
(Females used were nulliparous and non-pregnant)

Details on study design:

Study Design:
Initial consideration for exclusion of the test item was pH of ≤2.0 and ≥11.5.
The study was performed in two phases that is initial and confirmatory tests. Initial
test was performed with one animal. In the initial test, the test item did not cause any
irritant effects to the eye during observation period. Hence, confirmatory test was
conducted with additional two animals.

Test Procedure:
1. Pre-Instillation Procedure: Sixty minutes prior to test item instillation, tramadol hydrochloride injection
(2.5 mg/kg) was administered by subcutaneous injection (SC) to provide a therapeutic
level of systemic analgesia.
Five minutes prior to test item instillation, two drops/eye of a topical ocular
anaesthetic (0.5% tetracaine hydrochloride) was applied to both the eyes.
2. Instillation of Test Item: After pre- instillation procedure, test item was instilled into the conjunctival sac of the
left eye by gently pulling the lower lid away from the eye ball. Then the eye lids were
gently held together for about a second to prevent loss of test item. The right eye
remained untreated and served as the control.
The eye was rinsed using 0.9% w/v normal saline after 1 hour treatment (post
treatment).
3. Post-Instillation Procedure: The following post- instillation procedure was carried out as the test item caused
ocular lesions:
Eight hours (± 3 hours) after test item instillation, tramadol hydrochloride injection
(2.5 mg/kg) and meloxicam (0.5 mg/kg) was administered subcutaneously to provide
a continued therapeutic level of systemic analgesia.
After the initial 8-hours (± 3 hours) post-test item instillation, tramadol hydrochloride
injection (2.5 mg/kg) was administered subcutaneously 12 hours (± 3 hours), in
conjunction with meloxicam (0.5 mg/kg) every 24 hours (±3 hours), until the ocular
lesions resolve and no clinical signs of pain and distress are present.

Scoring of Ocular Lesions:
The treated and untreated eye of each animal was scored approximately at 1, 24, 48
and 72 hours after test item instillation. The examination of reactions (cornea, iris and
aqueous humor) was facilitated by use of slit lamp for both untreated and treated eyes
of the animals approximately at 24 and 48 hours for both initial and confirmatory test after test item instillation. The experiment was terminated after 72 hours observation for both initial and confirmatory test animals.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Scoring of Ocular Lesions:
In both initial and confirmatory test, treated eye (left) revealed occular lesions like
redness [Some blood vessels hyperaemic (injected)] at 1 and 24 hour observation. The
observed lesion reversed back to normal by 48 hour observation.
Refer Table 2
Slit Lamp Examination:
In both initial and confirmatory test, ocular lesion like conjunctivitis was observed at
24 hour observation. The observed lesions reversed back to normal by 48 hour
observation during slit lamp examination.
Refer Table 3

Other effects:

Clinical Signs of Toxicity and Mortality:
No clinical signs of toxicity and mortality were observed in both initial and
confirmatory test animals.
Body Weight:
No treatment related changes were noted in body weight and percent change in body
weight with respect to day 1 in both initial and confirmatory test. All the animals
revealed physiologically normal increase in body weight.

Any other information on results incl. tables

TABLE 2. INDIVIDUAL ANIMAL EYE IRRITATION/CORROSION SCORING RECORD

Initial Test                            Sex: Female                      Dose: 0.1 mL/animal                    Animal No.: Nc3679
Observation Period	Ocular Lesions
	Conjunctiva				Iris		Cornea
	Redness		Chemosis				Opacity		Area
Eyes	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
1 hr	1	0	0	0	0	0	0	0	-	-
24 hrs	1	0	0	0	0	0	0	0	-	-
48 hrs	0	0	0	0	0	0	0	0	-	-
72 hrs	0	0	0	0	0	0	0	0	-	-
Mean Tissue Score	0.33	0	0	0	0	0	0	0	-	-

Confirmatory Test                            Sex: Female                      Dose: 0.1 mL/animal       Animal No.: Nc3680
Observation Period	Ocular Lesions
	Conjunctiva				Iris		Cornea
	Redness		Chemosis				Opacity		Area
Eyes	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
1 hr	1	0	0	0	0	0	0	0	-	-
24 hrs	1	0	0	0	0	0	0	0	-	-
48 hrs	0	0	0	0	0	0	0	0	-	-
72 hrs	0	0	0	0	0	0	0	0	-	-
Mean Tissue Score	0.33	0	0	0	0	0	0	0	-	-

Confirmatory Test                            Sex: Female                      Dose: 0.1 mL/animal       Animal No.: Nc3681
Observation Period	Ocular Lesions
	Conjunctiva				Iris		Cornea
	Redness		Chemosis				Opacity		Area
Eyes	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
1 hr	1	0	0	0	0	0	0	0	-	-
24 hrs	1	0	0	0	0	0	0	0	-	-
48 hrs	0	0	0	0	0	0	0	0	-	-
72 hrs	0	0	0	0	0	0	0	0	-	-
Mean Tissue Score	0.33	0	0	0	0	0	0	0	-	-

hr: hour; hrs: hours; LE: Left Eye (Treated Eye); RE: Right Eye (Untreated/Reference control Eye)

Mean Tissue Score = (24 hr+48 hr+72 hr)/3

Redness:  0: Normal; 1: Some blood vessels hyperaemic (injected)

Chemosis: 0: Normal

Iris: 0: Normal                

Opacity: 0: No ulceration or opacity

 

TABLE 3. INDIVIDUAL ANIMAL SLIT LAMP EXAMINATION RECORD

Initial Test                    Sex: Female                        Dose: 0.1 mL/animal                          Animal No.: Nc3679
	Day 2 (24 hours)		Day 3 (48 hours)
Eye	LE	RE	LE	RE
Lids	13	N	N	N
Ducts	N	N	N	N
Cornea	N	N	N	N
Pupil	N	N	N	N
Sclera	N	N	N	N
Ciliary Bodies	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N

 

Confirmatory Test                    Sex: Female                        Dose: 0.1 mL/animal             Animal No.: Nc3680
	Day 2 (24 hours)		Day 3 (48 hours)
Eye	LE	RE	LE	RE
Lids	13	N	N	N
Ducts	N	N	N	N
Cornea	N	N	N	N
Pupil	N	N	N	N
Sclera	N	N	N	N
Ciliary Bodies	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N

Confirmatory Test                    Sex: Female                        Dose: 0.1 mL/animal             Animal No.: Nc3681
	Day 2 (24 hours)		Day 3 (48 hours)
Eye	LE	RE	LE	RE
Lids	13	N	N	N
Ducts	N	N	N	N
Cornea	N	N	N	N
Pupil	N	N	N	N
Sclera	N	N	N	N
Ciliary Bodies	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N

LE: Left Eye; RE: Right Eye; N: Normal/No Abnormality Detected; 13: Conjunctivitis

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the above results of the experiment and under the experimental conditions
employed, it is concluded that the mean score of the test item (2,4,8,10-Tetraoxa-3,9-
dithiaspiro [5.5]unde cane 3,3,9,9-tetraoxide) did not meet classification criteria and
hence not classified or categorized as per the Globally Harmonized System of
Classification and Labelling of Chemicals (GHS).

Executive summary:

The test item, (2,4,8,10-Tetraoxa-3,9-dithiaspiro [5.5]unde cane 3,3,9,9-tetraoxide) was evaluated for In vivo Eye Irritation/Serious Eye Damage in New Zealand White Rabbits.
The study was performed in two phases i.e., initial and confirmatory tests. Both the eyes of each experimental animal was examined within 24 hours before the treatment. The animals with absence of signs of eye irritation, ocular defects or pre-existing corneal injury were used for the treatment. 

The initial test was conducted using single female rabbit and confirmatory test was conducted using two female rabbits. The 0.1 mL (Initial test: 97.2 mg and Confirmatory test: 95.7 mg and 98.2 mg) of test item was instilled into the conjunctival sac of the left eye and right eye served as control. The eyes were scored approximately at 1, 24, 48 and 72 hours. Slit lamp examination was carried out using fluorescein strips and were scored approximately at 24 and 48 hour for both initial and confirmatory test.

All the animals (initial test and confirmatory test) were observed twice daily for clinical signs of toxicity and mortality. No treatment related clinical signs of toxicity and mortality were observed in all the animals (initial and confirmatory tests) after the test item instillation.
In both initial and confirmatory test, treated eye (left) revealed occular lesions like redness at 1 and 24 hour observation. The observed lesion reversed back to normal by 48 hour observation.

In both initial and confirmatory test, ocular lesion like conjunctivitis was observed at 24 hour observation. The observed lesions reversed back to normal by 48 hour observation during slit lamp examination.

The body weight was recorded on the day of receipt, on the day of treatment (prior to instillation of test item) and at termination of the experiment. No treatment related changes were noted in body weight and percent change in body we ight with respect to day 1 in both initial and confirmatory test. All the animals revealed physiologically normal increase in body weight in both initial and confirmatory tests.

For initial test and confirmatory test, the mean score calculated across 3 scoring times (approximately 24, 48 and 72 hours after test item instillation) for cornea, iris and conjunctival redness and conjunctival chemosis were 0, 0, 0.33 and 0 respectively.