Tetrahydrothiophene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrothiophene

EC Number:

203-728-9

EC Name:

Tetrahydrothiophene

Cas Number:

110-01-0

Molecular formula:

C4H8S

IUPAC Name:

tetrahydrothiophene

Details on test material:

Source: Pennwalt Holland
Batch: 271
Purity: >99%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent, England
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 175-177 for males and 141-144 for females- Fasting period before study:
- Housing: 5 of the smae sax to a cage in suspended polypropylene cages fitted with stainless steel mesh tops and floors
- Diet (e.g. ad libitum): Labsure diet LAD 1, Lavender Mill, Manea, Cambridgeshire, UK
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 10days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25
- Humidity (%): 28-80
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The absence of aerosol was demonstrated using an optical particle monitor (Royco Instruments Inc.) at each level of exposure during Weeks 1, 4, 8 and 12 of the study. Droplet numbers in test chambers were similar to those present in the control chamber. No appreciable amount of THT was present as droplets in the atmospheres generated.

Details on inhalation exposure:

The vapour of THT was administered to rats by whole-body exposure, 6 hours a day, 5 days a week for 13 weeks. Control rats received air only.

Vapour generation:
The vapour generation system, contained in a separate, sealed exposure chamber metered the liquid from a pressurised reservoir to a glass sinter, contained in a glass vessel through which air was passed. The vapour produced passed out of this vessel and into each chamber via an inlet duct. By varying the feed rate to each vapour generator it was possible to obtain the desired chamber concentrations.

Exposure chambers:
The exposure chambers were constructed of stainless steel and glass and were approximately 0.675 m3 in volume. The vapour from each generator entered the top of the chamber and was exhausted via a perforated plenum located in the base of each chamber. The exhaust from all chamber was drawn through an activated charcoal scrubbing system (Ventsorb R, Chemviron Ltd., Uppermill, Lancs.) by an extractor fan, before being vented to atmosphere. Extract flow was adjusted using gate valves downstream of the chamber to maintain a chamber internal pressure of 10 mm H20) below ambient, as indicated by magnehelic gauges connected to each chamber.
The rats were held during exposure in compartmented stainless steel mesh cages. A wet and dry bulb hygrometer was positioned in the chamber to monitor chamber temperature and relative humidity during exposure. Ports, fitted with removeable bungs, were present in the walls of each chamber to permit removal of chamber air samples for analysis. Routinely a port mid-centre of the chamber side wall was used.

Exposure chamber conditions:
- Measurement of chamber concentration of THT
The concentration of THT within the exposure chambers was determined on at least three occasions during each exposure.
- Air flow
The air flow into each chamber was monitored continuously using tapered-tube rotameters and recorded at 30-minute intervals throughout each exposure.
- Temperature
The air temperature in each chamber was monitored continuously with a mercury bulb thermometer and recorded at approximately hourly intervals throughout each exposure.
- Pressure
The air pressure in each exposure chamber, relative to that in the exposure room, was monitored using a magnehelic gauge and recorded at 30-minute intervals throughout exposure.
- Relative humidity
The humidity in each exposure chamber was monitored continuously with a wet and dry bulb hygrometer and recorded at hourly intervals throughout each exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of chamber air were withdrawn through glass tubes 10 cm in length, nominal bore 2 mm, packed for 2 - 3 cm of their length at one end only with chromosorb 102, 60 - 80 mesh. The vapour was adsorbed on this medium. The samples were thermally desorbed into a gas chromatograph and the amounts of THT collected determined by using external standards.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h/day; 5 d/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50, 275 and 1500 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
51, 236 and 1442 ppm (184, 852 and 5201 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

Post-exposure period: none

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

Clinical signs:
- During exposure
Clinical signs during exposure were recorded either as a group response where all visible animals appeared to be responding similarly, or as an individual response where one particular rat was affected.
- At other times
Animals were examined at least twice each day.

Bodyweight:
Each rat was weighed for allocation to groups and weekly thereafter from Week -1 (pre-exposure) until day of death.

Food consumption:
The quantity of food consumed by each cage of rats was recorded weekly, commencing at Week -1 (pre-exposure) until day of death.

Water consumption:
The amount of water consumed by each cage of rats was recorded daily, commencing at Week 3 until day of death.

Ophthalmoscopy:
The eyes of all rats were examined pre-exposure using a Keeler indirect ophthalmoscope. During Week 12 of exposure the eyes of Group 1 and 4 rats were examined.

Laboratory investigations:
Samples of blood were collected from 5 male and 5 female rats in each group on Week 5 of exposure. Samples of blood and urine were collected from all rats on Week 13 of exposure.
- Haematology Units
Packed cell volume (PCV) %
Haemoglobin (Hb) g/dl
Red cell count (RBC) 10e6/mm3
Mean corpuscular haemoglobin concentrations (MCHC)
calculated: Hb (g/dl) x 100 = PCV (%) %
Mean corpuscular haemoglobin (MCH) - calculated:
Hb (g/d1) x 100 = RBC (106/mm3) pg
Mean corpuscular volume (MCV) - calculated:
PCV (%) x 100 = RBC (10e6/mm3) fl
Reticulocyte counts (Retic) % (of red cells)
Total white cell count (WBC) 10e3/mm3
Differential count (Diff)
Neutrophils (N) 10e3/mm3
Lymphocytes (L) 10e3/mm3
Eosinophils (E) 10e3/mm3
Basophils (B) 10e3/mm3
Monocytes (M) 10e3/mm3
Platelet count (Plts) 10e3/mm3
Thrombotest (TT) s

- Blood biochemistry Units
Glucose mg/dl
Total protein g/dl
Albumin g/dl
Globulin g/dl
Albumin/Globulin ratio
Urea nitrogen mg/dl
Creatinine mg/dl
Alkaline phosphatase mU/ml
Glutamic-pyruvic transaminase (GPT) mU/ml
Glutamic-oxaloacetic transaminase (GOT) mU/ml
Total bilirubin mg/dl
Sodium (Na) mEq/l
Potassium (K) mEq/l
Calcium (Ca mEq/l
Inorganic phosphorus (P) mEq/l
Chloride (Cl) mEq/l
Cholesterol (Chol) mg/dl

- Urinalysis Units
Volume ml
pH
Osmolality (Osmol) mOsm/kg
Protein mg/dl
Qualitative tests:
Glucose, Ketones, Bile pigments, Haemoglobin

Sacrifice and pathology:

Sacrifice:
Following 13 weeks exposure all rats were sacrificed.

Macroscopic pathology and organ weight analysis:
A detailed macroscopic examination of all animals was performed and the following organs dissected free from each animal and weighed:
Lungs, Testes, Thyroids (with parathyroids), Epididymides, Liver, Prostate, Spleen, Ovaries, Adrenals, Brain, Heart, Pituitary, Kidneys

Microscopic examination:
Light microscope examination was performed on 5 µm-thick sections, stained with haematoxylin and eosin on the following tissues:
Nasal passages* (head for rostral and caudal nasal cavities), Adrenals*, Kidneys*, Oesophagus*, Urinary bladder*, Stomach*, Testes* (with epididymides), Trachea* (including bifurcation), Duodenum*, Jejunum*, Ovaries*, Lungs**, Ileum*, Uterus*, Tracheobronchial lymph nodes*, Caecum*, Brain* (section of medulla/pons, cerebellar cortex, cerebral cortex), Colon*, Salivary glands with attached mandibular lymph nodes*, Rectum*, Heart*, Aorta*, Pituitary*, Thyroids (with parathyroids)*, Lymph nodes* (cervical and mesenteric), Eyes*, Sciatic nerve*, Liver*, Thymus*, Sternum/ribs* (for bone and marrow), Spleen*, Skin*, Pancreas*, Spinal cord*, Gross abnormalities**, Optic nerve*.
* Groups 1 and 4 only
** All rats

Statistics:

All statistical analyses were carried out separately for males and females.

Data relating to food and water consumption were analysed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit.

Food consumption data were analysed using cumulative cage totals, and water consumption data were analysed as the total recorded intake over selected time periods. Bodyweight data were analysed using weight gains.

The following sequence of statistical tests were used for food consumption, water consumption, bodyweight, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
(ii) Bartlett's test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, logarithmic transformation was tried to see if a more stable variance structure could be obtained.
(iii) If no significant heterogéneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
(iv) Except for pre-dose data, analyses of variance were followed by Student's 't' test and Williams' test for a dose-related response, although only Williams' was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't' test and Williams' test (Shirley's test.

Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, the final bodyweight was used as a covariate in an attempt to allow for differences in bodyweight which might influence the organ weights.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CHAMBER ATMOSPHERE CONDITIONS
Chamber temperature:
Chamber temperatures during exposure were similar for all groups.

Chamber relative humidity:
Chamber relative humidities were similar for all groups.

CLINICAL OBSERVATIONS
Clinical signs:
During exposure; no abnormalities were seen for rats in Group 1 (Air Control).
Signs noted in exposed groups considered related to exposure to THT were as follows:
Groups 2: licking of the inside of the mouth, salivation on week 4 and lachrymation on week 7
Group 3: licking of the inside of the mouth, salivation on weeks 3, 4 and 5 and lachrymation on weeks 4 and 6
Group 4: licking of the inside of the mouth, salivation, lachrymation, adoption of a hunched posture, rubbing the chin on the grid mesh floor of the exposure cage and a marked reduced response to a sharp tap on the exposure chamber side wall.
At all other times; no abnormalities were seen.

Bodyweight:
Over the course of the 13-week study period there was a minimal reduction in bodyweight gain by female rats exposed to THT which attained statistical significance compared with control values. However, the reduced gain was not dose-related and therefore considered not of toxicological significance.
No treatment-related differences were seen for male rats exposed to THT.

Food consumption:
No treatment-related differences were seen.

Water consumption:
Measurement was initiated at the commencement of Week 3 of exposure after a difference in the amount of water consumed by High dose THT groups was noted visually.
Increased amounts of water were consumed by male and female rats in Group 4 (High dose THT) and by male rats in Group 3 (Inter. dose THT). These values were statistically significant compared with control values. Water consumption by female rats in Group 3 (Inter. dose THT) was also increased but did not achieve statistically significant separation compared with control data.

Ophthalmoscopy:
No treatment-related defects were seen.

LABORATORY INVESTIGATIONS
Haematology:
No differences were seen for male rats during Weeks 5 and 13 of the study.
Differences from control values achieving a degree of statistical significance were noted for female rats in Groups 3 (Inter. dose THT) and 4 (High dose THT) at Week 5, including:
reduced mean corpuscular haemoglobin concentration (MCHC)
(Group 4); increased mean corpuscular volume (MCV) (Groups 3 and 4); increased mean corpuscular haemoglobin (MCH) (Group 4); increased total white cell and lymphocyte counts (Group 4).
At Week 13 a similar effect was noted but confined to female rats in Group 4 only, including:
increased mean corpuscular volume; increased mean corpuscular haemoglobin; increased total white cell and lymphocyte counts.
The changes in red blood cell parameters MCHC, MCV and MCH were minimal and considered not be of toxicological significance.
The increases in total white cells and lymphocytes were inconsistent but the possibility of a treatment-related effect cannot be excluded.

Blood biochemistry:
Differences between treated and control groups achieving a degree of statistical significance during Weeks 5 and 13 of the study, were noted for; proteins, urea, creatinine, alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, electrolytes and cholesterol.
The differences were not dose-related, inconsistent between the sexes and minimal in scale, and were therefore considered not to be of toxicological significance.

Urinalysis:
There was a slight increase in the urinary pH of female rats from Group 4 (6.2) compared with control values (6.0). This difference was very small and considered unlikely to be of toxicological significance.
No other intergroup differences were seen achieving statistically significant separation.

TERMINAL STUDIES
Macroscopic pathology
No effects were seen that were considered related to exposure to THT.

Organ weights:
A minimal increase in the liver weights of female rats from Group 4 (High dose THT) achieved statistically significant separation from control values. However, this change was considered not of toxicological significance.
No other differences were seen that were considered related to exposure to THT.

Microscopic pathology:
Inhalation of THT by rats under the conditions of this study did not produce any observable treatment-related histopathological changes.
- Treatment-related changes
No histopathological changes were observed in any of the tissues examined that were considered to be related to treatment with THT.
- Incidental changes
The small number of minor histopathological changes observed in occasional tissues were within the background range normally observed in rats of this strain and age and were considered to be of no toxicological significance.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

>= 1 442 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (5201 mg/m3)

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEC

Remarks:

local irritation

Effect level:

51 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

other: (184 mg/m3)

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

236 ppm (analytical)

System:

eye

Organ:

lacrimal gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Bodyweights - group means (g)

	Group.and dosage
Week								-
	Male				Female
	Air Control	Low dose THT	Int.dose THT	High dose THT	Air Control	Low dose THT	Int.dose THT	High dose THT
Pre-dose
-1	201	202	199	203	157	156	159	157
Dosing 0	263	261	254	258	181	176	179	184
1	304	304	290	294	200	191	194	196
2	340	341	323	329	217	204	209	214
3	370	371	353	360	232	216*	223*	226*
4	400	397	377	392	247	225*	234*	237*
5	417	415	393	410	251	232*	237*	243*
6	447	439	414	436	262	239*	249*	251*
7	465	459	435	456	267	246*	253*	255*
8	483	477	451	472	275	253*	259*	261*
9	502	491	467	488	280	258*	265*	267*
10	518	506	481	507	286	264*	271*	273*
11	531	519	498	523	291	270	280	278
12	541	527	511	536	296	271*	277*	281*
13	544	529	509	536	292	269*	275*	279*
Gain -1 to 13	324	327	310	334	135	113*	115*	123*

* P<0.05 compared with control values using Williams' test

Water consumption - group mean cumulative values (g)

	Group and dosage
Week	Male				Female
	Air Control	Low dose THT	Int. dose THT	High dose THT	Air Control	Low dose THT	Int. dose THT	High dose THT
Dosing 3	202	207	228	281**	169	178	166	222*
4	403	409	458	569**	334	356	340	438*
5	617	620	689	860**	491	543	507	666*
6	836	827	922	1146**	655	719	684	900*
7	1047	1030	1153	1436**	816	901	850	1130**
8	1252	1228	1388	1728**	976	1079	1017	1355*
9	1460	1433	1629	2015**	1127	1262	1195	1584*
10	1664	1638	1879	2320**	1285	1453	1377	1811*
11	1867	1839	2127	2617**	1441	1636	1580	2027*
12	2062	2036	2374*	2908**	1587	1804	1759	2246*
13	2251	2230	2598*	3166**	1728	1983	1924	2437*

* P<0.05 compared with control data using Williams' test

** P<0.01 compared with control data using Williams' test

(Haematology – Female) Week 5

Group	PCV %	Hb g/dl	RBC Xl0^6/ mm^3	MCHC %	MCV fl	MCH pg	Retic %	WBC + Diff x10^3/mm^3						Plts x10^3/ mm^3	TT s
								Total	N	L	E	B	M
Air Control	48	17.2	7.9	35.6	62	21.9		9.5	1.30	8.08	0.11	0.00	0.03	1077	25
Low dose THT	48	16.8	7.6	35.4	62	22.0		11.5	1.47	10.01	0.04	0.00	0.00	1013	24
Int.dose THT	50	17.5	7.8	35.3	64**	22.4		10.6	0.62	9.95	0.07	0.00	0.00	1055	23
High dose THT	50	17.6	7.7	35.1*	65**	22.9**		15.2*	1.47	13.58*	0.14	0.00	0.05	1005	23

(Haematology - female) Week 13

Group	PCV %	Hb g/dl	RBC x10^6/ mm^3	MCHC %	MCV fl	MCH pg	Retic %	WBC + Diff x10^3/mm^3						Plts x10^3/ mm^3	TT s
								Total	N	L	E	B	M
Air Control	#48	#14.8	#7.3	30.8	66	20.3		7.9	1.12	6.70	0.05	0.00	0.01	839	20
Low dose THT	48	14.9	7.2	31.0	67	20.6		9.4	1.12	8.15	0.09	0.00	0.00	820	21
Int.dose THT	49	14.8	7.2	30.4	68	20.7		9.4	0.96	8.29	0.09	0.00	0.01	924	20
High dose THT	49	15.1	7.2	30.7	68*	21.0**		12.7**	1.28	11.31**	0.05	0.00	0.07	847	20

* P<0.05 compared with control data using Williams' test; ** P<0.01

# Distribution-free analysis applied to data F Frequency analysis applied to data

Applicant's summary and conclusion

Conclusions:

Clinical signs indicative of mild local irritation were seen at 236 and 1442 ppm. These signs were not apparent between exposures and not related to any macro-or microscopic abnormalities. Lacrimation and salivation occurred also once at the lowest concentration of 51 ppm However, this seems not significant due to the low frequency.
The amount of water consumed by male and female rats exposed at 1442 ppm and by male rats exposed at 236 ppm was significantly higher than control values but whithout any effects on blood chemistry, urinalysis and the histophatology of kidneys.
There were no other toxicologically significant effects at levels of exposure up to 1442 ppm.
The no effect level for clinical signs of irritation is 51 ppm.
The no effect level for sytemic toxicity is higher than 1442 ppm.

Executive summary:

Ten male and ten female Sprague-Dawley rats were whole-body exposed to analytical concentrations of 0, 51, 236 and 1442 ppm (184, 852 or 5201 mg/m³) tetrahydrothiophene (purity > 99%), six hours per day, five days per week for 13 weeks (Hardy, 1988). The study was performed according to the good laboratory practice and the OECD Test Guideline 413. The clinical symptoms of irritation (lacrimation, salivation, eyes closed) are a cluster of symptoms seen with increasing concentration. From 236 ppm, there was a more frequent occurrence of irritative symptoms (eyes closed). The significance of the symptoms of "licking the inside mouth and "shake his head" is unclear and questionable. The animals of the 1442 ppm group and the males of 236 ppm group drank more water than the animals in the control group. Ophthalmological examinations were unremarkable. In females of the 236 and 1442 ppm groups, the hematological parameters showed small differences compared to the control animals, but these changes were considered as not toxicologically significant, excepted an increase of white blood cells and lymphocytes at 1442 ppm. There was also a slight, not toxicologically significant increase in pH urine in females at 1442 ppm. Macroscopic examination and the determination of organ weights provided no abnormal findings. A minimal increase in liver weights was observed in females exposed to 1442 ppm but this effect was not considered toxicologically relevant. Histopathological examinations was performed on the lungs of all animals and on some other tissues of the control and 1442 ppm group animals. This included tissues from the nasal cavity (rostral and caudal). No histopathological changes were observed in any of the tissues examined that were considered to be related to treatment with THT. Free blood in sinuses was observed in one male animal of the 1442 ppm group and sinusoidal free blood in one female of the control group. These changes were considered as random. The NOAEC for the local irritation was 51 ppm (184 mg/m3) based on the clinical signs observed during exposure and the NOAEC for systemic effect was higher than 1442 ppm (5201 mg/m³).