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Description of key information

In the 90-day repeated dose oral toxicity study with Sulfolane, conducted according to the appropriate OECD 408 Test Guideline and in compliane with GLP, the study report concluded the NOEL level for females and males to be 25 mg/L and 100 mg/L drinking water, respectively, equivalent to 2.9 and 8.8 mg/kg bw/day (Huntingdon Life Sciences, 2001). The overall NOAEL is 2.9 mg/kg bw/day based on reduced immunological parameters in females that could be considered as treament related.

The subchronic oral (28-day gavage) NOAEL for sulfolane in rats is 200 mg/kg bw/day.  Kidney effects seen in male rats at this dose are considered to be indicative of alpha-2u-nephropathy, a male rat-specific effect that is not considered relevant to humans.  The subchronic inhalation (90-days) NOAEC for sulfolane in rats is 20 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Feb 2001 to 10 Aug 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, England
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 26-30 days on arrival and 39-43 days of age when study commenced.
- Weight at study initiation: 167-215 g (males) and 142-180 g (females)
- Fasting period before study: none
- Housing: 5 per sex in a stainless steel cages.
- Diet (e.g. ad libitum): The animals were allowed free access, except overnight before routine blood sampling, to an expanded rodent diet.
- Water (e.g. ad libitum): water from public supply, as libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: Each batch of diet was routinely analysed by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier's analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department of the Environment. Certificates of analysis were routinely received from the supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was prepared as a series of graded concentrations in tap water. Each formulation was prepared by dissolving an appropriate amount of Sulfolane with a small volume of vehicle and stirring by hand until a solution formed. Once the material was fully dissolved it was made up to the required final volume and mixed using a magnetic stirrer. Formulations were prepared weekly.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Water was used as vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced, the suitability of the proposed mixing procedure was determined by a trial preparation performed at the lowest highest concentrations (25 and 1600 mg/L). The stability of Sulfolane in the vehicle was assessed for storage at ambient temperature (nominally 21 DC) for 4 and 8 days and in a refrigerator (nominally 40C) for 4, 8 15 days. Analysis at ambient temperature for 15 days was scheduled to be carried out but this was not perforned in error. This deviation did not affect the integrity of the study. In addition, samples of each formulation prepared for administration in Weeks l, 6 and 12 of treatment were analysed for achieved concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, continuous
Dose / conc.:
25 mg/L drinking water
Dose / conc.:
100 mg/L drinking water
Dose / conc.:
400 mg/L drinking water
Dose / conc.:
1 600 mg/L drinking water
Dose / conc.:
2.1 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 25 mg/L
Dose / conc.:
8.8 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 100 mg/L
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 400 mg/L
Dose / conc.:
131.7 mg/kg bw/day (actual dose received)
Remarks:
males: equivalent to 1600 mg/L
Dose / conc.:
2.9 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 25 mg/L
Dose / conc.:
10.6 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 100 mg/L
Dose / conc.:
42 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 400 mg/L
Dose / conc.:
191.1 mg/kg bw/day (actual dose received)
Remarks:
females: equivalent to 1600 mg/L
No. of animals per sex per dose:
10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not selected
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): random
Positive control:
Not used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked included: sings of ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week a more detailed physical examination was performed.

BODY WEIGHT: Yes
- Time schedule for examinations: The bodyweight of each animal was recorded during the acclimatisation period, on the day that treatment commenced (Week O), at weekly intervals during the treatment period and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes. Group mean food conversion efficiencies were calculated for each week of treatment.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The amount of water supplied to each cage and that remaining recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before commencement of treatment both eyes of all animals were examined by means of an indirect ophthalmoscope, after the instillation of 0.5% tropicamide. The structures exarnined included the following: adnexa, conjunctivae, cornea and sclera, anterior chamber and iris (pupil dilated), lens and vitreous, ocular fundus. During Week 13 of treatment, all animals from Groups 1 and 5 were similarly examined.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 13 of treatment.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 13 of treatment.
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals were examined before treatment and on a weekly basis thereafter.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity, startle reflex, pupil reflex, touch response, approach response, body temperature, body weight, landing footsplay, tail pinch response, tighting reflex

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No 2)

HISTOPATHOLOGY: Yes (see table No 2)
Statistics:
The significance of inter-group differences in hematology and blood chemistry was first assessed by Bartlett's test. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the Control group, incorporating adjustment for multiple comparisons where necessary.
For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used. Inter-group differences in macroscopic and microscopic pathology were assessed using Fishers Exact test. Unless stated, group mean values or incidences for the treated groups were not significantly different
from those of the Controls (p>0.05).
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs related to treatment were observed.
Mortality:
no mortality observed
Description (incidence):
No animals died during the treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight gain in the first week of treatment was lower in high dose (1600 mg/L) animals than the control group. Bodyweight gain thereafter was unaffected, resulting in similar overall bodyweight gain. Bodyweight gain in the rest of the groups was unaffected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected in any of the treatment groups.
Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion efficiency was slightly low, compared with Controls, during Week I in animals receiving 1600 mg/L. Thereafter food conversion efficiency was unaffected in these animals resulting in similar overall food conversion efficiency.
Food conversion efficiency in animals receiving 25, 100 or 400 mg/L was not affected by treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was unaffected.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related effects observed in any of the animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological examination in Week 13 revealed low lymphocyte, monocyte and large unstained cell counts, compared with the controls, in females receiving 100, 400 or 1600 mg/L, resulting in a concomitant reduction in the total white blood cell count in these animals. Though the effect was greatest in females receiving 1600 mg/L, overall there was no strong trend with administered concentration of Sulfolane. There was no similar finding in males. There was no evidence of any chronic inflammatory change or of compromised immune function in females, or any effect upon bone marrow, thymus or spleen that would account for the reduced numbers of these leucocytes. Other inter-group differences that attained statistical significance were considered minor or lacked dosage relationship and so were considered to represent normal biological variation. Such differences included the slightly prolonged prothrombin times in males receiving 1600 mg/L, slightly high mean cell volumes and reduced activated partial thromboplastin times in females receiving 1600 mg/L, slightly low basophil counts in females receiving 100 mg/L and in animals receiving 400 or 1600 mg/L and slightly low large unstained cell counts in males receiving 400 or 1600 mg/L which was attributed to high values in two control animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical examination of the plasma in Week 13 revealed, compared with the Controls, slightly low alanine amino-transferase activities and slightly high creatinine concentrations in males receiving 1600 mg/L. The cause of the slight reduction of plasma alanine amino-transferase activities in males receiving 1600 mg/L was not established in this study. Toxicological significance is usually associated with increased plasma alanine amino-transferase activities and therefore this finding is not considered biologically significant.
Other inter-group differences that attained statistical significance such as the variations in sodium concentrations were minor or lacked dosage relationship and so were considered to represent normal biological variation. Such changes included, in males receiving 1600 mg/L, low aspartate amino-transferase activities, which was attributed to a raised mean value for the controls due to unusually high values in two animals and reduced sodium concentration which was attributed to a markedly low value in one animal.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related changed in the in hand observations. Salivation was observed throughout the treatment in all dose groups, including control group. Behaviour in the arena was unaffected by treatment. No treatment-related effects were noted during manipulation. No treatment-related changes in motor activity was noticed in any of the animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in organ weights in any of the animals.
Absolute and bodyweight relative uterus weights were higher than Controls in females receiving 100 or 1600 mg/L; however these changes were considered to represent the slightly increased incidence of uterine fluid distension, due to the stage of oestrus in some of these animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic changes were noted in any of the animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The only findings considered related to treatment were seen in the kidneys of males given Sulfolane at 400 mg/L or above. There was an increase in the incidence and severity of cortical tubules with hyaline droplets, compared with controls, in males receiving 400 or 1600 mg/L. This is related to the toxicological syndrome known as "hydrocarbon nephropathy" which results from an accumulation of alpha-2µ-globulin in the cortical tubules of affected kidneys. This finding is specific to the male rat and is therefore of no toxicological significance to man. There was an increase in the incidence and severity of cortical tubular basophilia in males receiving 1600 mg/L. The incidence of granular casts of the medulla was slightly high in males receiving 1600 mg/L.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/L drinking water
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: equivalent to 8.8 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
Remarks on result:
other: equivalent to 2.9 mg/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/L drinking water
System:
immune system
Organ:
other: reduction in immunological parameters
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

See attachment for result tables.

Conclusions:
In the 90-day repeated dose oral toxicity study with Sulfolane, conducted according to the appropriate OECD 408 Test Guideline and in compliane with GLP, it was concluded the NOEL level for females and males to be 25 mg/L and 100 mg/L, respectively. The overall NOAEL is 2.9 mg/kg bw/day based on reduced immunological parameters in females.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2.9 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
immune system
Organ:
blood

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
20 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a 90-day repeated oral toxicity study, conducted according to an appropriate OECD 408 Test Guideline and in compliance with GLP, groups of 10 male and 10 female CD rats received Sulfolane continuously in the drinking water for 13 weeks at concentrations of 25, 100, 400 or 1600 mg/L drinking water. A similarly constituted control group received tap water only (Huntingdon Life Sciences, 2001).

No deaths were observed during the treatment period and there were no clinical signs of toxicity related to the treatment. Bodyweight gain and food conversion efficiency were low during the first week in animals treated with 1600 mg/L, but appeared normal thereafter. Food and water consumption were unaffected by treatment. The mean overall achieved dosages were 2.1, 8.8, 35.0 and 131.7 mg/kg/day for males and 2.9, 10.6, 42.0 and 191.1 mg/kg/day for females receiving 25, 100, 400 and 1600 mg/L, respectively. There were no treatment related ocular findings. The functional observational battery of tests did not indicate any findings indicative of neurotoxicity. Haematological examination in Week 13 revealed low lymphocyte, monocyte and large unstained cell counts, compared with the controls, in females receiving 100, 400 or 1600 mg/L, resulting in a concomitant reduction in the total white blood cell count in these animals. Biochemical examination of the plasma in week 13 revealed, when compared with the controls, slightly low alanine amino-transferase activities and slightly high creatinine concentrations in males receiving 1600 mg/L drinking water.

There were no organ weight changes or macroscopic findings that were attributed to treatment with Sulfolane. Histopathological examination revealed findings in the kidneys of males receiving 400 or 1600 mg/L comprising cortical tubules with hyaline droplets in males receiving 400 or 1600 mg/L and cortical tubular basophilia and granular casts of the medulla in males receiving 1600 mg/L.

It is concluded that the continuous daily administration of Sulfolane via the drinking water to CD rats for 13 weeks at concentrations up 1600 mg/L was well-tolerated. Hydrocarbon nephropathy occurred in males given 400 mg/L which is a result from alpha-2µ-globulin accumulation in kidneys. Changes to lymphocyte, monocyte and large unstained cell counts were evident in females given 100 mg/L or more. The no-observed-effect level (NOEL) was considered to be 100 mg/L for males (equivalent to 8.8 mg/kg/day) and 25 mg/L for females (equivalent to 2.9 mg/kg/day). The overall NOAEL is 2.9 mg/kg bw/day based on reduced immunological parameters in females.

The study does not include urinalysis which is not a standard requirement under OECD Test Guideline 408. The study includes the full histpathological examinationand and it goes beyond the standard test requirements to assess neurotoxicity (via functional observation battery) and ophthalmology. There is detailed information on the alpha-2µ-globulin histopathology, heamatology and biochemical parameters and the effects observed are related to hydrocarbon induced nephropathy, the absence of urinalysis is not critical for the validity and interpretation of the results. There would be no added value in repeating the study to include this parameter.

Male and female rats were given oral doses of 0, 60, 200 or 700 mg/kg sulfolane by gavage for 28 days (MHW, 1996). An additional group of animals were given either 0 or 700 mg/kg sulfolane for 28 days followed by a 14-day recovery period. There was reduced body weight gain and food consumption in males and females of the 700 mg/kg dose group. Kidney effects were seen in the male rats at doses of 200 and 700 mg/kg sulfolane which appear to be indicative of alpha-2u-nephropathy, a male rat-specific effect that is not considered relevant to humans. The NOAELs for male and female rats is 200 mg/kg/day.

Exposure of male and female rats to sulfolane vapor for 90 to 110 days at concentrations of 2.8, 4.0 or 20.0 mg/m3 did not induce systemic toxicity (Andersen, 1977). There were no treatment-related effects on body weight, hematology, clinical chemistry or histopathology. A NOAEC of 20 mg/m3 was established. Guinea pigs were exposed by inhalation to sulfolane either as a vapor/aerosol (20, 159 or 200 mg/m3) or as a vapor (2.8 and 4.0 mg/m3) for 90-110 days. At 200 mg/m3, animals exhibited chronic pleuritis, leukopenia (decreased white blood cells), and fatty vacuolization of the liver. At 159 mg/m3 or lower, there were no treatment-related effects. A NOAEC of 159 mg/m3 was established.

In the 28-day repeat oral dosing study with a 14 -day recovery period, kidney effects were seen in the male rats at doses of 200 and 700 mg/kg sulfolane which appear to be indicative of alpha-2u-nephropathy, a male rat-specific effect that is not considered relevant to humans. In the repeat inhalation studies, the NOAECs for rats and guinea pigs were 20 and 159 mg/m3, respectively. For the purposes of risk assessment, the lower of the two NOAEC values (20 mg/m3) was used to derive DNELs.

Justification for classification or non-classification

There are sufficient data available on sulfolane to conclude that classification is not warranted under GHS/CLP.