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

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 July 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 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted on 07 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

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

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Rattus norvegicus

Sex:

male/female

Details on test animals or test system and environmental conditions:

Body Weight Range at Receipt:
Sighting Study: I- Males : 182.47 g to 196.26 g, Females: 161.07 g to 164.82 g
II- Males : 189.36 g to 195.32 g, Females: 162.81 g to 178.75 g
Main Study: Males : 182.52 g to 196.28 g, Females: 162.59 g to 178.99 g
The body weights for each sex were within ±20% of the mean body weight of all previously exposed animals.
No. of Animals/ Group:
Sighting study - I and II - 3 males and 3 females each
Main Study - 5 males and 5 females per group
The animals were randomly selected and marked for individual identification.
(Females used were nulliparous and non-pregnant)
Age at Treatment: 09 to 10 weeks
Acclimatization period: All the animals were identified by tail marking using a red permanent marker pen. Additionally, a cage card was displayed which included study no., cage no., sex, animal no. (temporary), start date and end date of acclimatization period.
Treatment period: The last 4 digits of the animal number was marked on the tail using a black permanent marker pen and additionally, a cage card was displayed which included study no., animal no. (permanent), sex, dose, cage no., treatment date and date of necropsy.

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 for sighting study I 19.7°C to 22.6°C and relative humidity 46% to 66%, for sighting study II 19.7°C to 22.9°C and relative humidity 49% to 65% and for main study 19.8°C to 22.7°C and relative humidity 44% to 66% with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
b. Housing:
Maximum of three animals of same sex were housed in a standard polycarbonate cage (size: L 430 × B 280 × H 210 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water bottle fitted with stainless steel sipper tube. Clean sterilized corncob was provided as bedding material.
c. Feed:
Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period (except during restraining and exposure 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 (except during the restraining and exposure period).

Acclimatization:
Healthy young adult animals were acclimatized for a minimum period of five days to laboratory conditions prior to treatment and were observed for clinical signs once daily. During the acclimatization period all the animals were restrained for one hour (±10 minutes) in the restraining tubes on two different days and observed for one hour (±10 minutes) during and post restraining. Veterinary examinations of all the animals was performed on the day of receipt of animals.

Grouping:
The animals taken (15 males and 15 females) for the experiment were weighed and arranged in ascending order of their body weights. These body weight stratified rats were distributed to all the experimental groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ±20% (male: -15.20% to +6.88% and female: -9.62% to +7.79%) of the mean body weight of each sex. The grouping was done prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out any statistically significant difference between groups within each sex.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 4 µm

Geometric standard deviation (GSD):

>= 1.5 - <= 3

Remark on MMAD/GSD:

Sighting Study I: MMAD (µm) 2.84, GSD 2.85
Sighting Study II: MMAD (µm) 2.77, GSD 2.88
Main Study G1: MMAD (µm) 2.72, GSD 2.91
Main Study G2: MMAD (µm) 3.10, GSD 2.89
Main Study G3: MMAD (µm) 3.09, GSD 2.82

Details on inhalation exposure:

Inhalation Exposure System:
Inhalation exposure was conducted using a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA, with a provision of at least 12 air changes per hour. The exposure unit consisted of stackable exposure tiers with top and bottom sections or plates for introduction and exhaust of test item. Each tier has 12 exposure ports which were used for exposing up to 10 animals and sampling of test atmosphere. All parts (except O-ring seals) were constructed of stainless steel. The volume of each inner plenum of inhalation chamber was 0.76 liters (11 cm diameter and 8 cm height) that consisted of total 12 port hole (one tier). Each tier was made of an inner plenum and an outer plenum that were connected to each other through rectangular trumpets (tubes) and connector cones. The chamber was equipped with a bottom inlet section (plate) and a top exhaust section. Both sections were designed to connect easily to other equipment through swivels. The animals were restrained in the transparent polycarbonate restraining tubes. The chamber was made to sit on a rotating table that allows easy access to observe all animals at each port during exposure. This unit ensured that uniform distribution of test item that provided continuous “fresh” test item to each animal and precluded re-breathing the exhaled air. The chamber provided continuous supply of fresh aerosol to all the restrained animals through the trumpet from inner plenum of chamber. A slight positive pressure was maintained in inner plenum of chamber. The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only dynamic inhalation exposure chamber. The whole inhalation chamber was situated inside the fume hood. The outlet air from chamber exhaust was treated with 1% w/v sodium hydroxide solution and passed through absorbent cotton before being evacuated into the atmosphere.

Test Aerosol Generation:
The Rotating Brush Generator (Palas RBG 1000) was used to generate the dust particles (aerosols) into an airborne state. The rotating brush was situated inside the dispersion head and below the brush, the cylindrical powder reservoir located which was filled with the test item to disperse the test item inside the chamber inner plenum. The transportation piston pushed the compact mass of test item by adjusting the rotation of brush to 600 RPM at the feed rate and air flow rate of 20 L/min with pressure 60 psi to generate the target concentration of test item. The outlet of rotating brush generator was connected to the bottom inlet section (plate) through swivels to the inner plenum. The generated dust aerosols were discharged from the top of exposure chamber.

Exposure System Monitoring:
The chamber temperature, relative humidity, oxygen and carbon dioxide concentration were measured during exposure at an empty port of the exposure chamber. The air flow meter (rotameter) was used to regulate and measure, inlet and outlet air of the chamber.

Determination of Particle Size Distribution:
Particle size distribution was determined gravimetrically by using a 7 stage Cascade Mercer Impactor supplied by In-Tox Products, USA. Based on the results of mass deposited on every stage, Mass Median Aerodynamic Diameters (MMAD) and Geometric Standard Deviations (GSD) were calculated using Microsoft Excel Sheet. The target range for the Mass Median Aerodynamic Diameter was 1 to 4 µm and GSD was 1.5 to 3.0. During sampling, the Impactor air flow rate was 1.52 L/min for one minute. The particle size was measured three times during exposure period at animal breathing zone after equilibration period (i.e. ± 15 minutes from breathing zone concentration sampling).
The cumulative mass less than the stated cut of diameter versus particle size (effective cut of diameter) on log probability scale was plotted to determine MMAD and GSD.
The mean MMAD and GSD were 2.84 µm and 2.85 and 2.77 µm and 2.88 for sighting study I and II respectively.
During main study, the mean MMAD and GSD were found to be 2.72, 3.10, 3.09 µm and 2.91, 2.89, 2.82 respectively.

Relative Humidity, Temperature, Oxygen and Carbon Dioxide Concentration:
The IAQ probe (Indoor Air Quality probe) supplied by Gray Wolf Sensing Solutions, Ireland, was used for continuous monitoring of Relative humidity, temperature, oxygen and carbon dioxide content in the chamber and recorded four times during exposure (i.e., ± 10 minutes) after equilibration period. The mean chamber conditions like temperature, relative humidity, oxygen concentration and carbon dioxide concentration recorded during the exposure period are as follows and found within the acceptable range.

Exposure Air Flow Rate:
Air flow through the generation system into the chamber was controlled and monitored using flow-meter (rotameter) and outlet of the chamber air flow was controlled by rotameter. The actual flow rate of chamber inlet was 20 L/min with 60 psi pressure and outlet air from chamber was 15 L/min maintained throughout the exposure period. The air inlet flow rate was recorded four times during exposure period (i.e., ± 10 minutes).

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Remarks on duration:

Animals were exposed to aerosolized test item continuously for four hours after five minutes of equilibration period of the chamber concentration.

Concentrations:

Sighting study I was performed with 3 male and 3 female animals at the concentration of 1.22 mg/L of air and 100% mortality was observed in sighting study I, hence sighting study II was conducted with additional 3 male and 3 female animals at the concentration of 0.54 mg/L to determine the range of lethal concentrations for main study. Clinical signs and mortality was observed during the sighting study II.
Main study was performed with three different concentration levels. Each group consisted of 5 males and 5 females. The doses were selected for main study based on outcome of the sighting study II. G1 (High dose): 0.12 mg/L of air, G2 (Low dose): 0.052 mg/L of air, G3 (Mid dose): 0.080 mg/L of air.

The nominal concentration was calculated according to “mass of test item disseminated into the exposure system during the generation period divided by the total airflow through the inhalation chamber during the same time period”.
Sighting study I: 6.15 mg/L, Sighting study II: 4.29 mg/L
G1 (High dose): 3.17 mg/L, G2 (Low dose): 2.49 mg/L, G3 (Mid dose): 2.77 mg/L

Breathing zone concentration (actual aerosol concentration) was determined gravimetrically using a 47 mm filter paper loaded in an in-line sampling device. Actual aerosol concentration was determined by gravimetric method. The 1.52 L/min critical orifice was used to draw the air from the inhalation chamber for one minute at animal breathing zone. It was carried out three times during exposure [i.e. 60 minutes (±15 minutes), 120 minutes (±15 minutes) and 180 minutes (±15 minutes)] after equilibration period for each exposure day.
The mean breathing zone concentration (actual concentration) was 1.22 and 0.54 mg/L of air for sighting study I and II respectively, whereas the mean breathing zone concentration (actual concentration) during main study for group G1, G2 and G3 was found to be 0.12, 0.052 and 0.080 mg/L of air respectively.

No. of animals per sex per dose:

Sighting study I: 1.22 mg/L of air, 3 males and 3 females
Sighting study II: 0.54 mg/L of air, 3 males and 3 females
Main Study
G1 (High dose): 0.12 mg/L of air, 5 males and 5 females per group
G2 (Low dose): 0.052 mg/L of air, 5 males and 5 females per group
G3 (Mid dose): 0.080 mg/L of air, 5 males and 5 females per group

Control animals:

no

Details on study design:

Technical Pre-Test:
A technical pre-test, without animals was conducted to assess and establish the feasibility of achieving the following objectives:
• The recommended test atmosphere and stability.
• Target concentration (5 mg/L of air) or maximum attainable concentration for limit test.
• Target particle size (MMAD of 1 to 4 µm) with a geometric standard deviation (1.5 to 3.0).
The technical pre-test was carried out without animals. As such test item was used during technical pre-test to generate the dust aerosols through rotating brush generator. During the technical pre-test for sighting study I, maximum attainable concentration i.e. 1.22 mg/L of air was achieved at the feed rate of 030 mm/hour with a rotation of 600 RPM. The mean breathing zone concentration achieved at this stage was 1.22 mg/L of air and the MMAD and GSD obtained were 2.66 µm and 2.74 respectively which were in the prescribed range. The achieved concentration i.e. 1.22 mg/L of air at the feed rate of 030 mm/hour with a rotation of 600 RPM was considered as the maximum achievable concentration. Therefore, for sighting study I, to achieve the target concentration the rotating brush generator feed rate was maintained at 030 mm/hour with a rotation of 600 RPM.
During technical pre-test for sighting study II, maximum attainable concentration i.e. 0.55 mg/L of air was achieved at the feed rate of 007 mm/hour with a rotation of 600 RPM. The mean breathing zone concentration achieved at this stage was 0.55 mg/L of air and the MMAD and GSD obtained were 2.87 µm and 2.92 respectively which were in the prescribed range. The achieved concentration i.e. 0.55 mg/L of air at the feed rate of 007 mm/hour with a rotation of 600 RPM was considered as the maximum achievable concentration. Therefore, for sighting study II, to achieve the target concentration the rotating brush generator feed rate was maintained at 007 mm/hour with a rotation of 600 RPM.
During technical pre-test for main study (G1), maximum attainable concentration i.e. 0.12 mg/L of air was achieved at the feed rate of 003 mm/hour with a rotation of 600 RPM. The mean breathing zone concentration achieved at this stage was 0.12 mg/L of air and the MMAD and GSD obtained were 2.73 µm and 2.97 respectively which were in the prescribed range. The achieved concentration i.e. 0.12 mg/L of air at the feed rate of 003 mm/hour with a rotation of 600 RPM was considered as the maximum achievable concentration. Therefore, for main study (G1), to achieve the target concentration the rotating brush generator feed rate was maintained at 003 mm/hour with a rotation of 600 RPM.
During technical pre-test for main study (G2), maximum attainable concentration i.e. 0.053 mg/L of air was achieved at the feed rate of 001 mm/hour with a rotation of 600 RPM. The mean breathing zone concentration achieved at this stage was 0.053 mg/L of air and the MMAD and GSD obtained were 3.04 µm and 2.87 respectively which were in the prescribed range. The achieved concentration i.e. 2.87 mg/L of air at the feed rate of 001 mm/hour with a rotation of 600 RPM was considered as the maximum achievable concentration. Therefore, for main study (G2), to achieve the target concentration the rotating brush generator feed rate was maintained at 001 mm/hour with a rotation of 600 RPM.
During technical pre-test for main study (G3), maximum attainable concentration i.e. 0.080 mg/L of air was achieved at the feed rate of 002 mm/hour with a rotation of 600 RPM. The mean breathing zone concentration achieved at this stage was 0.080 mg/L of air and the MMAD and GSD obtained were 2.94 µm and 2.87 respectively which were in the prescribed range. The achieved concentration i.e. 0.080 mg/L of air at the feed rate of 002 mm/hour with a rotation of 600 RPM was considered as the maximum achievable concentration. Therefore, for main study (G3), to achieve the target concentration the rotating brush generator feed rate was maintained at 002 mm/hour with a rotation of 600 RPM.

Treatment of Exhaust Air:
Exhaust air was treated with 1% w/v sodium hydroxide solution and passed through absorbent cotton before evacuated into the atmosphere.

Clinical Signs and Mortality:
All the animals were observed for clinical signs and pre-terminal deaths at 1 hr (± 10 mins), 2 hrs (± 10 mins), 3 hrs (± 10 mins) and 4 hrs (± 10 mins) during exposure period. Post exposure, clinical signs and pre-terminal deaths were observed at 30 to 40 minutes and 1 hour (± 10 mins), and once daily thereafter for clinical signs and twice daily for mortality during the 14 days post exposure period. The clinical signs observations included but not limited to changes in skin, fur, eyes, mucous membrane, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern.
Body Weight:
Individual animal body weight was recorded at receipt, prior to exposure (Day 1) and on day 2, 4, 8 and 15 during the experimental period. The body weight of dead animals during the observation period were also recorded.
Necropsy:
At the end of the observation period, all surviving animals were euthanized by intraperitoneal administration of sodium thiopentone and subjected to necropsy and complete gross pathological examination. The dead animals were also subjected to complete gross pathological examination.

Statistics:

The particle size (MMAD and GSD) was calculated using Microsoft Excel Sheet. The LC50 of each sex i.e. males and females were calculated using Finney Probit Analysis.
LC50=0.091, Upper limit=0.096, Lower limit=0.086.

Results and discussion

Preliminary study:

Clinical Signs and Mortality:
In sighting study I, the animals were exposed at mean concentration of 1.22 mg/L of air determined by gravimetric method. During exposure no clinical sign was noted until one hour observation, at second hour observation all the animals showed clinical signs like nasal bleeding. During third hour observation tremors along with nasal bleeding was noted in all the animals. At fourth hour observation three male and two female animals were found dead and surviving one female animal showed nasal bleeding and tremors however at post exposure 30-40 minutes and 1 hour observation remaining one female animal showed tremors, nasal bleeding, hyperactivity and was found dead.
In sighting study II, the animals were exposed at mean concentration of 0.54 mg/L of air determined by gravimetric method. During exposure no clinical signs were noted, until second hour observation. At third hour observation all animals showed clinical signs like nasal bleeding. At fourth hour observation one male and three female rats showed nasal bleeding and tremors, the remaining two male rats showed nasal bleeding followed by found dead was noted. At post exposure 30-40 minutes all animals showed clinical signs like nasal bleeding and tremors and also one male and two female animals were found dead. During post exposure 1 hour observation remaining one female animal showed clinical signs like tremors, nasal bleeding and followed by mortality.
Body Weight:
During sighting study I & II 100% mortality was noted on the day of exposure, hence dead animal body weights were taken.
Pathology:
No gross pathology lesions were observed in terminal sacrificed animals, however in dead animals externally nasal discharge, red was observed in sighting study I and sighting study II animals and internally lung discoloration, red to reddish was observed during sighting study I and II.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.091 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

In group G1 (High dose), the animals were exposed at mean concentration of 0.12 mg/L of air. During exposure no clinical signs noted until second hour observation at third hour observation one male and one female animal was found dead and remaining all animals showed clinical signs like nasal bleeding which was persisted until fourth hour observation, At post exposure 30-40 minutes and 1 hour observation one male and one female animal showed nasal bleeding and remaining all animals showed nasal bleeding, tremors. On day 2 observation, three males and three female animals were found dead remaining one male and one female animal showed hunched back. On day 3 observation the exhibited clinical signs reversed back to normal throughout the experimental period.
In group G2 (Low dose), the animals were exposed at mean concentration of 0.052 mg/L of air. During exposure no clinical signs were noted, however post exposure i.e., 30 – 40 minutes and 1hour observation one male and three female animals showed clinical signs like hunched back. On day 2 observation the exhibited clinical sign animals reversed back to normal throughout the experimental period.
In group G3 (Mid dose), the animals were exposed at mean concentration of 0.080 mg/L of air. During exposure no clinical signs were noted, however post exposure i.e., 30-40 minutes all animals showed clinical signs like hunched back in addition to this two male and two female rats showed tremors and at 1 hour post exposure observation all surviving animals showed hunched back along with this five males and three female animals showed tremors and in addition two male and two female rats showed nasal bleeding. On day 2 observation, two male and two female animals were found dead, and remaining all animals showed hunched back which was persisted until day 3 observation. On day 4 observation the exhibited clinical sign animals reversed back to normal and all the animals were normal throughout the experimental period.

Clinical signs:

observations of tremors

Remarks:

Refer Tables 1 and 2

Body weight:

During main study decrease in body weight and percent change in body weight were observed in all the survived animals on day 2 and showed increase in body weight on day 4, 8 and 15.
Refer Tables 3 and 4

Gross pathology:

In main study no gross pathology lesions were observed in terminal sacrificed animals, however in dead animals externally no abnormality detected for all G1 and G3 group animals internally lung discoloration, red was observed in G1 group animals.
Refer Tables 12 & 13

Any other information on results incl. tables

• TABLE 1. INDIVIDUAL ANIMAL CLINICAL SIGNS AND MORTALITY RECORD OF SIGHTING STUDY

Group  & Concentration (mg/L of Air)	Animal No.	Sex	Day 1						Days
			During Exposure				Post exposure
			1 hr*	2 hrs*	3 hrs*	4 hrs*	30-40 min	1 hr*	2	3	4	5	6	7	8	9	10	11	12	13	14	15
Sighting Study-I & 1.22	Rh2623	M	N	74	74,44	D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh2624	M	N	74	74,44	D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh2625	M	N	74	74,44	D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh2626	F	N	74	74,44	74,44	74,44, 24	74,44,24 D	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh2627	F	N	74	74,44	D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh2628	F	N	74	74,44	D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Sighting Study-II & 0.54	Rh4356	M	N	N	74	74,44	74,44,D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh4357	M	N	N	74	74,D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh4358	M	N	N	74	74,D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh4359	F	N	N	74	74,44	74,44,D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh4360	F	N	N	74	74,44	74,44,D	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Rh4361	F	N	N	74	74,44	74,44	74,44,D	-	-	-	-	-	-	-	-	-	-	-	-	-	-

• TABLE 2. INDIVIDUAL ANIMAL CLINICAL SIGNS AND MORTALITY RECORD OF MAIN STUDY

Group  & Concentration (mg/L of Air)

Animal No.

Sex

Day 1

Days

During Exposure

Post exposure

1 hr*

2 hrs*

3 hrs*

4 hrs*

30-40 min

1 hr*

2

3

4

5

6

7

8

9

10

11

12

13

14

15

G1 & 0.12

Rh4362

M

N

N

74

74

74

74

7

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4363

M

N

N

D

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4364

M

N

N

74

74

74,44

74,44

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4365

M

N

N

74

74

74,44

74,44

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4366

M

N

N

74

74

74,44

74,44

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4367

F

N

N

74

74

74

74

7

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4368

F

N

N

74

74

74,44

74,44

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4369

F

N

N

D

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4370

F

N

N

74

74

74,44

74,44

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4371

F

N

N

74

74

74,44

74,44

D

-

-

-

-

-

-

-

-

-

-

-

-

-

G2 & 0.052

Rh4372

M

N

N

N

N

7

7

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4373

M

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4374

M

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4375

M

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4376

M

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4377

F

N

N

N

N

7

7

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4378

F

N

N

N

N

7

7

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4379

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4380

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rh4381

F

N

N

N

N

7

7

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Group  & Concentration (mg/L of Air)

Animal No.

Sex

Day 1

Days

During Exposure

Post exposure

1 hr*

2 hrs*

3 hrs*

4 hrs*

30-40 min

1 hr*

2

3

4

5

6

7

8

9

10

11

12

13

14

15

G3 & 0.080

Rh4382

M

N

N

N

N

7,44

7,44,74

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4383

M

N

N

N

N

7

7,44

7

7

N

N

N

N

N

N

N

N

N

N

N

N

Rh4384

M

N

N

N

N

7,44

7,44,74

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4385

M

N

N

N

N

7

7,44

7

7

N

N

N

N

N

N

N

N

N

N

N

N

Rh4386

M

N

N

N

N

7

7,44

7

7

N

N

N

N

N

N

N

N

N

N

N

N

Rh4387

F

N

N

N

N

7

7

7

7

N

N

N

N

N

N

N

N

N

N

N

N

Rh4388

F

N

N

N

N

7

7

7

7

N

N

N

N

N

N

N

N

N

N

N

N

Rh4389

F

N

N

N

N

7,44

7,44,74

D

-

-

-

-

-

-

-

-

-

-

-

-

-

Rh4390

F

N

N

N

N

7

7,44

7

7

N

N

N

N

N

N

N

N

N

N

N

N

Rh4391

F

N

N

N

N

7,44

7,44,74

D

-

-

-

-

-

-

-

-

-

-

-

-

-

 N: Normal; M: Male; F: Female; min: minute; hr(s): hour(s); -: Not applicable; D: Dead; 44: Tremors; 74: Nasal bleeding; 7: Hunched back *: ± 10 minutes

• TABLE 3. INDIVIDUAL ANIMAL BODY WEIGHT (g) AND PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 DURING SIGHTING STUDY

Group &    Concen-tration (mg/L of Air)	Animal No.	Sex	Body Weight (g)					Percent Change in Body Weight with Respect to Day 1				Dead Animal Body Weight (g)
			Day 1	Day 2	Day 4	Day 8	Day 15	Day 1-2	Day 1-4	Day 1-8	Day 1-15
Sighting Study-I & 1.22	Rh2623	M	201.04	-	-	-	-	-	-	-	-	200.02
	Rh2624	M	216.98	-	-	-	-	-	-	-	-	216.12
	Rh2625	M	213.30	-	-	-	-	-	-	-	-	212.45
	Mean		210.44	-	-	-	-	-	-	-	-	-
	±SD		8.35	-	-	-	-	-	-	-	-	-
	n		3	-	-	-	-	-	-	-	-	-
	Rh2626	F	175.66	-	-	-	-	-	-	-	-	175.12
	Rh2627	F	179.49	-	-	-	-	-	-	-	-	179.02
	Rh2628	F	176.87	-	-	-	-	-	-	-	-	175.68
	Mean		177.34	-	-	-	-	-	-	-	-	-
	±SD		1.96	-	-	-	-	-	-	-	-	-
	n		3	-	-	-	-	-	-	-	-	-
Sighting Study-II &    0.54	Rh4356	M	219.36	-	-	-	-	-	-	-	-	217.63
	Rh4357	M	216.41	-	-	-	-	-	-	-	-	214.19
	Rh4358	M	213.92	-	-	-	-	-	-	-	-	211.86
	Mean		216.56	-	-	-	-	-	-	-	-	-
	±SD		2.72	-	-	-	-	-	-	-	-	-
	n		3	-	-	-	-	-	-	-	-	-
	Rh4359	F	186.23	-	-	-	-	-	-	-	-	185.24
	Rh4360	F	180.46	-	-	-	-	-	-	-	-	179.63
	Rh4361	F	194.83	-	-	-	-	-	-	-	-	193.21
	Mean		187.17	-	-	-	-	-	-	-	-	-
	±SD		7.23	-	-	-	-	-	-	-	-	-
	n		3	-	-	-	-	-	-	-	-	-

• TABLE 4. INDIVIDUAL ANIMAL BODY WEIGHT (g) AND PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 DURING MAIN STUDY

Group &    Concen-tration (mg/L of Air)	Animal No.	Sex	Body Weight (g)					Percent Change in Body Weight with Respect to Day 1				Dead Animal Body Weight (g)
			Day 1	Day 2	Day 4	Day 8	Day 15	Day 1-2	Day 1-4	Day 1-8	Day 1-15
G1 & 0.12	Rh4362	M	213.84	211.05	215.38	230.43	354.22	-1.30	0.72	7.76	65.65	-
	Rh4363	M	219.32	-	-	-	-	-	-	-	-	218.50
	Rh4364	M	221.52	-	-	-	-	-	-	-	-	219.40
	Rh4365	M	225.98	-	-	-	-	-	-	-	-	223.74
	Rh4366	M	260.10	-	-	-	-	-	-	-	-	258.21
	Mean		228.15	211.05	215.38	230.43	354.22	-1.30	0.72	7.76	65.65	-
	(±)SD		18.39	-	-	-	-	-	-	-	-	-
	n		5	1	1	1	1	1	1	1	1	-
	Rh4367	F	178.77	176.49	180.02	189.65	205.96	-1.28	0.70	6.09	15.21	-
	Rh4368	F	189.34	-	-	-	-	-	-	-	-	187.53
	Rh4369	F	192.72	-	-	-	-	-	-	-	-	190.22
	Rh4370	F	194.67	-	-	-	-	-	-	-	-	192.35
	Rh4371	F	209.76	-	-	-	-	-	-	-	-	206.91
	Mean		193.05	176.49	180.02	189.65	205.96	-1.28	0.70	6.09	15.21	-
	(±)SD		11.18	-	-	-	-	-	-	-	-	-
	n		5	1	1	1	1	1	1	1	1	-
G2 & 0.052	Rh4372	M	217.61	215.04	219.49	234.32	259.32	-1.18	0.86	7.68	19.17	-
	Rh4373	M	219.91	216.40	221.86	235.43	261.15	-1.60	0.89	7.06	18.75	-
	Rh4374	M	225.39	222.57	228.32	244.61	268.68	-1.25	1.30	8.53	19.21	-
	Rh4375	M	239.38	237.92	241.68	254.94	278.49	-0.61	0.96	6.50	16.34	-
	Rh4376	M	241.32	240.04	243.92	257.22	282.53	-0.53	1.08	6.59	17.08	-
	Mean		228.72	226.39	231.05	245.30	270.03	-1.03	1.02	7.27	18.11	-
	(±)SD		11.01	11.86	11.23	10.65	10.29	0.45	0.18	0.84	1.32	-
	n		5	5	5	5	5	5	5	5	5	-
	Rh4377	F	177.67	176.98	179.39	189.36	205.16	-0.39	0.97	6.58	15.47	-
	Rh4378	F	188.31	185.66	189.43	196.08	214.82	-1.41	0.59	4.13	14.08	-
	Rh4379	F	206.04	204.75	208.51	219.92	238.95	-0.63	1.20	6.74	15.97	-
	Rh4380	F	198.17	197.83	199.99	208.59	225.72	-0.17	0.92	5.26	13.90	-
	Rh4381	F	200.07	198.01	201.21	211.13	229.38	-1.03	0.57	5.53	14.65	-
	Mean		194.05	192.65	195.71	205.02	222.81	-0.72	0.85	5.65	14.82	-
	(±)SD		11.16	11.14	11.38	12.22	13.11	0.50	0.27	1.06	0.89	-
	n		5	5	5	5	5	5	5	5	5	-

Group &    Concen-tration (mg/L of Air)	Animal No.	Sex	Body Weight (g)					Percent Change in Body Weight with Respect to Day 1				Dead Animal Body Weight (g)
			Day 1	Day 2	Day 4	Day 8	Day 15	Day 1-2	Day 1-4	Day 1-8	Day 1-15
G3 & 0.080	Rh4382	M	213.38	-	-	-	-	-	-	-	-	210.64
	Rh4383	M	225.58	223.02	226.17	236.29	261.38	-1.13	0.26	4.75	15.87	-
	Rh4384	M	225.27	-	-	-	-	-	-	-	-	221.36
	Rh4385	M	239.94	236.41	240.54	251.12	277.49	-1.47	0.25	4.66	15.65	-
	Rh4386	M	258.15	256.53	259.76	270.51	295.33	-0.63	0.62	4.79	14.40	-
	Mean		232.46	238.65	242.16	252.64	278.07	-1.08	0.38	4.73	15.31	-
	(±)SD		17.17	16.87	16.85	17.16	16.98	0.42	0.21	0.07	0.79	-
	n		5	3	3	3	3	3	3	3	3	-
	Rh4387	F	183.05	181.49	183.96	190.32	209.76	-0.85	0.50	3.97	14.59	-
	Rh4388	F	191.51	189.82	192.05	199.73	217.82	-0.88	0.28	4.29	13.74	-
	Rh4389	F	192.44	-	-	-	-	-	-	-	-	189.39
	Rh4390	F	202.63	200.96	203.76	208.36	227.93	-0.82	0.56	2.83	12.49	-
	Rh4391	F	205.65	-	-	-	-	-	-	-	-	203.43
	Mean		193.06	190.76	193.26	199.47	218.50	-0.85	0.45	3.70	13.61	-
	(±)SD		10.79	9.77	9.96	9.02	9.10	0.03	0.14	0.77	1.06	-
	n		5	3	3	3	3	3	3	3	3	-

M: Male; F: Female; SD: Standard Deviation; -: Not applicable; n: No. of animals

• TABLE 12. INDIVIDUAL ANIMAL GROSS PATHOLOGICAL FINDINGS DURING SIGHTING STUDY

Group & Concentration (mg/L of Air)	Animal No.	Sex	Fate	Gross Pathological Findings
				External	Internal
Sighting Study-I & 1.22	Rh2623	M	D	Nasal discharge,red	Lung-Discoloration, Red
	Rh2624	M	D	Nasal discharge,red	Lung-Discoloration, Red
	Rh2625	M	D	Nasal discharge,red	Lung-Discoloration, Red
	Rh2626	F	D	Nasal discharge,red	Lung-Discoloration, Red
	Rh2627	F	D	Nasal discharge,red	Lung-Discoloration, Red
	Rh2628	F	D	Nasal discharge,red	Lung-Discoloration, Red
Sighting Study-II & 0.54	Rh4356	M	D	Nasal discharge,red	Lung-Discoloration, Reddish
	Rh4357	M	D	Nasal discharge,red	Lung-Discoloration, Reddish
	Rh4358	M	D	Nasal discharge,red	Lung-Discoloration, Reddish
	Rh4359	F	D	Nasal discharge,red	Lung-Discoloration, Reddish
	Rh4360	F	D	Nasal discharge,red	Lung-Discoloration, Reddish
	Rh4361	F	D	Nasal discharge,red	Lung-Discoloration, Reddish

• TABLE 13. INDIVIDUAL ANIMAL GROSS PATHOLOGICAL FINDINGS DURING MAIN STUDY

Group & Concentration (mg/L of Air)	Animal No.	Sex	Fate	Gross Pathological Findings
				External	Internal
G1 & 0.12	Rh4362	M	TS	NAD	NAD
	Rh4363	M	D	NAD	NAD
	Rh4364	M	D	NAD	Lung-Discoloration, Red
	Rh4365	M	D	NAD	Lung-Discoloration, Red
	Rh4366	M	D	NAD	Lung-Discoloration, Red
	Rh4367	F	TS	NAD	NAD
	Rh4368	F	D	NAD	Lung-Discoloration, Red
	Rh4369	F	D	NAD	Lung-Discoloration, Red
	Rh4370	F	D	NAD	Lung-Discoloration, Red
	Rh4371	F	D	NAD	Lung-Discoloration, Red
G2 & 0.052	Rh4372	M	TS	NAD	NAD
	Rh4373	M	TS	NAD	NAD
	Rh4374	M	TS	NAD	NAD
	Rh4375	M	TS	NAD	NAD
	Rh4376	M	TS	NAD	NAD
	Rh4377	F	TS	NAD	NAD
	Rh4378	F	TS	NAD	NAD
	Rh4379	F	TS	NAD	NAD
	Rh4380	F	TS	NAD	NAD
	Rh4381	F	TS	NAD	NAD
G3 & 0.080	Rh4382	M	D	NAD	NAD
	Rh4383	M	TS	NAD	NAD
	Rh4384	M	D	NAD	NAD
	Rh4385	M	TS	NAD	NAD
	Rh4386	M	TS	NAD	NAD
	Rh4387	F	TS	NAD	NAD
	Rh4388	F	TS	NAD	NAD
	Rh4389	F	D	NAD	NAD
	Rh4390	F	TS	NAD	NAD
	Rh4391	F	D	NAD	NAD

• NAD: No Abnormalities Detected; M: Male; F: Female; TS: Terminal Sacrifice; D: Dead

Applicant's summary and conclusion

Interpretation of results:

Category 2 based on GHS criteria

Conclusions:

Under the experimental conditions employed and based on the results of the experiment, it is concluded that LC50 of the test item, “(2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]unde cane 3,3,9,9-tetraoxide)” was 0.091 mg/L of air respectively when administered by nose-only inhalation route through a single four hours exposure to rats. The test item was classified under “Category 2 (0.05

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 acute inhalation toxicity in Sprague Dawley rats.

The objective of the study was to assess the toxic potential and to determine the LC₅₀ of test item, (2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]unde cane 3,3,9,9-tetraoxide) when administered by inhalation route through flow-past nose-only dynamic inhalation equipment for a single 4 hours exposure to rats. Three male and three female Sprague Dawley rats were used for each sighting study (I and II). For main study 5 males and 5 females Sprague Dawley rats were used each for group (G1: High Dose; G2: Low Dose and G3: Mid Dose). 

The test item was triturated with mortar and pestle, the same test item was used during technical pre-test, sighting study and main study to generate the dust aerosols through rotating brush generator. The technical pre-test was carried out without animals. Based on the results of technical pretest, to achieve the target concentration the rotating brush generator feed rate was maintained.

The mean chamber conditions like temperature, relative humidity, oxygen concentration, carbon dioxide concentration, MMAD and GSD were observed during the exposure and were found within the acceptable range.

All the animals were observed for clinical signs and pre-terminal deaths at 1 hr (±10 mins), 2 hrs (±10 mins), 3 hrs (±10 mins) and 4 hrs (±10 mins) during exposure and 30 to 40 minutes and 1 hour (±10 mins) post-exposure on day 1 and once daily thereafter for clinical signs and twice daily for mortality till 14 days post exposure period. Individual animal body weight was recorded on day 1 (on the day of exposure) prior to the exposure and on day 2, 4, 8 and 15. During exposure period, the chamber (exposure) conditions were recorded. All survived animals were euthanized after 14 days post exposure period by intraperitoneal administration of sodium thiopentone and the gross pathological findings were recorded.

The LC₅₀ was calculated by Finney probit analysis method for combined sex.

In sighting study I, the animals were exposed at mean concentration of 1.22 mg/L of air determined by gravimetric method. During exposure no clinical sign was noted until one hour observation, at second hour observation all the animals showed clinical signs like nasal bleeding. During third hour observation tremors along with nasal bleeding was noted in all the animals. At fourth hour observation three male and two female animals were found dead and surviving one female animal showed nasal bleeding and tremors however at post exposure 30-40 minutes and 1 hour observation remaining one female animal showed tremors, nasal bleeding, hyperactivity and was found dead.

In sighting study II, the animals were exposed at mean concentration of 0.54 mg/L of air determined by gravimetric method. During exposure no clinical signs were noted, until second hour observation. At third hour observation all animals showed clinical signs like nasal bleeding. At fourth hour observation one male and three female rats showed nasal bleeding and tremors, the remaining two male rats showed nasal bleeding followed by found dead was noted. At post exposure 30-40 minutes all animals showed clinical signs like nasal bleeding and tremors and also one male and two female animals were found dead. During post exposure 1 hour observation remaining one female animal showed clinical signs like tremors, nasal bleeding and followed by mortality.

Based on the sighting study - II results, main study animals were exposed to three different concentrations i.e. 0.12, 0.052 and 0.080 mg/L of air determined by gravimetric method for group G1 (High dose), G2 (low dose) and G3 (mid dose), respectively.

In group G1 (High dose), the animals were exposed at mean concentration of 0.12 mg/L of air, determined by gravimetric method. During exposure no clinical signs noted until second hour observation at third hour observation one male and one female animal was found dead and remaining all animals showed clinical signs like nasal bleeding which was persisted until fourth hour observation. At post exposure 30-40 minutes and 1 hour observation one male and one female animal showed nasal bleeding and remaining all animals showed nasal bleeding, tremors. On day 2 observation, three males and three female animals were found dead remaining one male and one female animal showed hunched back. On day 3 observation the exhibited clinical signs reversed back to normal throughout the experimental period.

In group G2 (Low dose), the animals were exposed at mean concentration of 0.052 mg/L of air, determined by gravimetric method. During exposure no clinical signs were noted, however post exposure i.e., 30 – 40 minutes and 1hour observation one male and three female animals showed clinical signs like hunched back. On day 2 observation the exhibited clinical sign animals reversed back to normal throughout the experimental period.

In group G3 (Mid dose), the animals were exposed at mean concentration of 0.080 mg/L of air determined by gravimetric method. During exposure no clinical signs were noted, however post exposure i.e., 30-40 minutes all animals showed clinical signs like hunched back in addition to this two male and two female rats showed tremors and at 1 hour post exposure observation all surviving animals showed hunched back along with this five males and three female animals showed tremors and in addition two male and two female rats showed nasal bleeding. On day 2 observation, two male and two female animals were found dead, and remaining all animals showed hunched back which was persisted until day 3 observation. On day 4 observation the exhibited clinical sign animals reversed back to normal and all the animals were normal throughout the experimental period.

During sighting study I & II  100% mortality was noted on the day of exposure, hence dead animal body weights were taken.

During main study decrease in body weight and percent change in body weight were observed in all the survived animals on day 2 and showed increase in body weight on day 4, 8 and 15.

No gross pathology lesions were observed in terminal sacrificed animals, however in dead animals externally nasal discharge, red was observed in sighting study I and sighting study II animals internally lung discoloration, red to reddish was observed during sighting study I and II.

In main study no gross pathology lesions were observed in terminal sacrificed animals, however in dead animals externally no abnormality detected for all G1 and G3 group animals internally lung discoloration, red was observed in G1 group animals.