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Repeated dose toxicity: inhalation

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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, OECD 413 Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test compound: Dimethylsulfoxyde
CAS no.: 67-68-5
Source: Sigma Aldrich
Batch : 29356-089
Purity : > 99%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Supplier: Charles River (UK) Limited, Manson Road Margate, Kent
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: males ca. 290 g, females ca.200g
- Housing: 5/cage (suspended stainless steel
- Diet: SDS Rat and Mouse no. 1 SQC modified maintenance diet, Special Diets Services, Witham, Essex
- Water: ad libitum tap water
- Fasting period before study: no
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21+/-2°C
- Relative humidity: 55+/-10 %
- Light/Dark cycle (hours): 12/12
- Air changes (per hr): no data

IN-LIFE DATES: 18 October 1999 to 15 February 2000

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Mean achieved droplet size characteristics for the treatrnent period at the concentration of 2.783 mg/l:
MMAD sigma-g Inhalable traction
(µm EAD) (% <7.0 (µm EAD)
5.8 2.20 59
EAD Equivalent aerodynamic diamete
Details on inhalation exposure:
The rats were exposed to the control/test atmosphere in 50L ADG snout-only exposure chambers. Each chamber was housed in a separate extract cabinet to prevent potential cross-contamination between groups.

The liquid test material was delivered to a concentric jet atomiser and generated as vapour only, or a mixture of vapour and droplets, into a stream of heated air for administration to the rats, by inhalation from snout only exposure chambers. The target chamber concentrations were achieved by metering the test substance from polypropylene syringes mounted on syringe drivers. The atmospheres produced by the atomisers were further diluted with air to give the final chamber concentrations of DMSO.

The flow of air to each concentric jet atomiser was calibrated using precision made tapered glass tube flowmeters and was monitored throughout the exposures using in-line flowmeters. The settings of the test substance metering system required to obtain the larget chamber concentrations were determined during the preliminary phase of the study, based on the result of Gas-liquid Chromatographie (GC) analysis of atmosphere samples.

It was predicted (based upon observations during a previous inhalation toxicity study performed at these laboratories - Huntingdon Life Sciences Report No. EFA 019/982007 - that DMSO would be present entirely as vapour at the target concentrations for the Low and Intemediate dose groups in this study. Samples collected from the atmospheres generated during the preliminary trials at the Low and Intermediate concentrations confimed that the test material would be present only as vapour. At the High chamber concentration, the DMSO was found to be present as a mixture of vapour and droplets. Based on these findings, no droplet size deteminations were performed during the main study for atmospheres generated at the Low and Intemediate target concentrations. At the High concentration, the aerodynamic behaviour of the droplet component of the atmospheres was detemined, at pre-detemined points during the exposure period, using a cascade impactor.

The usage of DMSO was recorded, on each day of treatment, for each of the three test groups. The nominal chamber concentration was calculated from these data and the calibrated airtlow for each chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations during the study were confimed by analysis using Gas Chromatography.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours a day, 7 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.310, 0.964, 2.783 mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
10 males and 10 females.
An additional 10 male and 10 female rats concurrently exposed at the Control and High dose levels were retained following the final exposure for a further 4 weeks of withdrawal (recovery) to assess the reversibility of any adverse findings.
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: 4 weeks (control and high dose group)
Positive control:
Not appropriate

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment and then weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined : Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before study and on week 13
- Dose groups that were examined:control group and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:on week 13
- Animals fasted: No
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine:on week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of exposure, during Week 12 and during the recovery period. A shortened battery of observations was conducted during each of Weeks 1-11 of the study.
- Dose groups that were examined: all

OTHER: oestrus cycle (Vaginal smears were prepared daily from all female rats during weeks 8 and 9 of the study)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (Adrenals, Lungs : all lobes and mainstem bronci, Brain : medulla, cerebellar and cortical sections, Ovaries, right Epididymides, Spleen, Heart, Testes, Kidneys, Thymus where present, Liver)

HISTOPATHOLOGY: Yes
Histopathological examinations were performed on all scheduled tissues (marked with *) for Groups control and high dose, and on tissues from all groups (marked with X). These tissues were embedded in paraffin wax and sections 4 - 5 µm thick were cut, processed and stained with haematoxylin and eosin for examination by light microscopy.
Sections, approximately 2 µm, were cut from the testes (transverse sections) and epididymides (longitudinal sections) and stained with PAS-haematoxylin.

Adrenals*, Heart*, Sciatic nerve*, Alimentary tract, Kidneys*, Seminal vesicles*, Oesophagus*, Larynx*, Skeletal muscle*, Stomach (antrum, glandular and non glandular)*, Duodenum*, Liver*, Skin, Lungs (all lobes and mainstem bronchi)X, Spinal column, Spinal cord (cervical, thoracic and lumbar)*, Lymph nodes (cervical, mesenteric and tracheobronchial)*, Jejunum*, Ileum*, Spleen*, Caecum*, Sternum*, Colon*, Mammary gland, Testes*, Rectum*, Nasal passages (head for rostral and caudal nasal cavities)*, Thymus (where present)*, Animal identification mark, Thyroids (with parathyroids)*, Aorta*, Optic nerve, Tongue, Brain (4 levels)*, Ovaries*, Trachea (including bifurcation)*, Right epididymides*, Pancreas*, Eyes*, Pharynx*, Ureter, Femur with joint (for bone marrow in situ), Pituitary*, Urinary bladder*, Prostate Uterus (corpus and cervix)*, Gross abnormalitiesX, Salivary gland* Vagina

The lymph nodes were identified separately. The remaining head was retained for paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth, eyelids, lacrymal gland, Harderian gland and Zymbal's gland.
Other examinations:
SEMINOLOGY on left Epididymis immediately following sacrifice, samples from all males were taken for:
- Sperm analysis: Sperm samples taken from vas deferens (from left side) from rats from all groups were assessed for motility using a computer assisted sperm analyser (CASA).
- Morphology: A manual assessment of sperm morphology was performed
- Cauda epididymis (from left side): The cauda epididymis was weighed and homogenised and the number of sperm was counted using a computer assisted sperm analyser (CASA).
Statistics:
All statistical analyses were carried out separately for males and females.
Food consumption was analysed using cage mean values
For all other parameters the analyses were carried out using the individual animal as the experimental unit. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data.
If the data consist 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:
Bartlett's test was applied to test for heterogeneity of variance between treatments; where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), and more than two groups were being compared, group means were compared using Williams' test for a dose-related response (Williams, 1971-72), or if there was evidence for a non-monotonic response, Dunnett's test (Dunnett, 1955, 1964). For separate two-group comparisons, a Student's t test was used.
If significant heterogeneity of variance was present (and could not be removed by a logarithmic transformation), groups were compared using Shirley's non-parametric test for a dose-related response (Shirley, 1977), or if there was evidence for a non-monotonic response, Dunn's test (Dunn 1964). For separate two-group comparisons, a Wilcoxon rank sum test (Wilcoxon 1945) was used.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
A Group 4 male was sacrificed on humane grounds in Week 13 due to the condition of the upper incisors and subsequent weight loss.
Treatment-related clinical signs consisted of red staining around the nose, observed post-exposure in a proportion of Group 3 (Intermediate dose) from Week 5. All Group 4 (High dose) rats were observed to have red staining around the nose pre and post exposure from Week 4 of the study period, continuing throughout the exposure period. Other clinical observations were consistent with exposure to a mildly irritant atmosphere.

BODY WEIGHT AND WEIGHT GAIN
Rats exposed to DMSO gained less weight over the 13 weeks of exposure compared with controls, statistically significant when exposed at 2.783 mg/L. (-17% in males and -16% in females). The trend was reversed during the recovery phase of the study. The differences were small and the absence of any other indications of systemic toxicity were considered likely to be related to a degree of inapetance caused by the mildly irritant nature of the test atmosphere. This was supported by small concomitant reductions in food consumption.

FOOD CONSUMPTION
Differences between Control and Treated groups were minimal and of no toxicological importance.

WATER CONSUMPTION
Differences between Control and Treated groups were minimal and of no toxicological importance.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related differences between the groups.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS
There were no differences between control and test groups considered to be attributable to exposure to DMSO.

NEUROBEHAVIOUR
Treatment with dimethylsulphoxide (DMSO) for 13 weeks followed by a four-week recovery period was not associated with any behavioural changes that were considered indicative of neurotoxicity.

ORGAN WEIGHTS
The lung weights of male rats exposed to DMSO were significantly greater than Control weights however, the difference was small, not dose related and not seen in females. The difference is considered not to be attributable to exposure to DMSO.
There were no other differences between the groups that were considered to be attributable to exposure to DMSO.

GROSS PATHOLOGY
There were no findings that were considered to be attributable to exposure to DMSO.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological findings were consistent with exposure to a mildly irritant atmosphere.
Treatment related changes were found in the nasal passages and pharynx of High dose males and females, which were killed after treatment for 90 days. These changes were not found in rats from the Low and Intermediate dose groups killed at this time.

Treatment-related changes in the nasal passages of High dose rats comprised lesions in the inferior ventral medial meatus (pseudogland formation in the respiratory epithelium and epithelial hyperplasia with or without inflammation in the squamous epithelium), and an increased degree of eosinophilic inclusions in the olfactory epithelium. In the pharynx, prominent goblet cells were present in the majority of High dose rats.

In rats killed after the recovery period, changes were still evident in the nasal passages of High dose male and females and also in the pharynx of the females.

OTHER FINDINGS
Oestrus cycle: There were no differences between control and test groups considered to be attributable to exposure to DMSO.
Seminology: There were no differences between control and test groups considered to be attributable to exposure to DMSO.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
2.783 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: No exposure-related toxicity
Dose descriptor:
NOAEC
Remarks:
Local respiratory irritation
Effect level:
0.964 mg/L air (analytical)
Sex:
male/female
Dose descriptor:
LOAEC
Remarks:
local respiratory irritation
Effect level:
2.783 mg/L air (analytical)
Basis for effect level:
other: Irritation of the nasal passages

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Group Study Mean concentration (mg/l)
   Nominal Analysed
2 (Low dose) 0,3 0,31
3 (Inter dose) 1 0,964
4 (High dose) 3 2,793

Applicant's summary and conclusion

Conclusions:
The no adverse effects concentration could be established at 0.964 mg/l for respiratory tract irritation and 2.783 mg/l for systemic toxicity.
Executive summary:

The potential toxicity of Dimethylsulfoxide (DMSO) was evaluated following repeated inhalation administration for 13 weeks, according to OECD Guideline and US EPA OPPTS and in compliance with GLP. Three groups of rats (10 males and 10 females) of the Crl:CD®BR strain were exposed to a vapour or vapour/liquid droplet atmosphere generated from pure (100%) DMSO, 6 hours a day, 7 days a week for 13 weeks using a snout only exposure system. A fourth group, acting a control was exposed to air only. An additional 10 male and 10 female rats concurrently exposed at the Control and High dose levels were restrained following the final exposure for the further 4 weeks of withdrawal (recovery) to assess the reversibility of any adverse findings.

The mean study analysed chamber concentrations were 0.310, 0.964 and 2.793 mg/l.

Clinical signs both during exposures and at other times were monitored and recorded. Body weight and food consumption were recorded weekly and water consumption was recorded daily. All main and withdrawal groups rats were subjected to ophtalmoloscopic examination prior to the start of exposures, rats from the control and high dose group were examined during week 13.

A functional observation battery was conducted in all groups. Comprehensive observations were conducted prior to the start of exposure, during week 12 and during the recovery period. A shortened battery of observations was conducted during each of weeks 1-11 of the study. Laboratory investigations (haematology, biochemical parameters, urinalysis) were conducted on week 13 in rats from all main groups.

The oestrus cycle of female rats was monitored. Following sacrifice rats were submitted to detailed macroscopic examinations followed by preservation of tissues and subsequent histopathological examination. Male rats were submitted to seminological investigations. Immunotoxicological investigations were carried out on groups of satellite rats. The humoral immune response to the T-cell dependant antigen, sheep red blood cells was assessed using a plaque cell-forming assay.

A Group 4 male was sacrificed on humane grounds in Week 13 due to the condition of the upper incisors and subsequent weight loss.

Treatment-related clinical signs consisted of red staining around the nose, observed post-exposure in a proportion of Group 3 (Intermediate dose) from Week 5. All Group 4 (High dose) rats were observed to have red staining around the nose pre and post exposure from Week 4 of the study period, continuing throughout the exposure period. Other clinical observations were consistent with exposure to a mildly irritant atmosphere.

Rats exposed to DMSO gained less weight over the 13 weeks of exposure compared with controls, statistically significant when exposed at 2.783 mg/L. (-17% in males and -16% in females). The trend was reversed during the recovery phase of the study. The differences were small and the absence of any other indications of systemic toxicity were considered likely to be related to a degree of inapetance caused by the mildly irritant nature of the test atmosphere. This was supported by small concomitant reductions in food consumption.

Differences between Control and Treated groups on food consumption, water consumption were minimal and of no toxicological importance. There were no treatment-related differences on ophtalmoscopic examinations between the groups. There were no differences on haematology, cluinical chemistry and urinalysis between control and test groups considered to be attributable to exposure to DMSO.

Treatment with dimethylsulphoxide (DMSO) for 13 weeks followed by a four-week recovery period was not associated with any behavioural changes that were considered indicative of neurotoxicity.

The lung weights of male rats exposed to DMSO were significantly greater than Control weights however, the difference was small, not dose related and not seen in females. The difference is considered not to be attributable to exposure to DMSO. There were no other differences between the groups that were considered to be attributable to exposure to DMSO. There were no findings on gross pathology that were considered to be attributable to exposure to DMSO. Histopathological findings were consistent with exposure to a mildly irritant atmosphere. Treatment related changes were found in the nasal passages and pharynx of High dose males and females, which were killed after treatment for 90 days. These changes were not found in rats from the Low and Intermediate dose groups killed at this time. Treatment-related changes in the nasal passages of High dose rats comprised lesions in the inferior ventral medial meatus (pseudogland formation in the respiratory epithelium and epithelial hyperplasia with or without inflammation in the squamous epithelium), and an increased degree of eosinophilic inclusions in the olfactory epithelium. In the pharynx, prominent goblet cells were present in the majority of High dose rats. In rats killed after the recovery period, changes were still evident in the nasal passages of High dose male and females and also in the pharynx of the females. There were no differences on oestrus cycle or seminology between control and test groups considered to be attributable to exposure to DMSO. Cli

nical and histopathological observations were consistent with exposure to a mildly irritant atmosphere. There were no other clinical observations, haematology, blood chemistry, urinalysis or reprotoxic findings that were treatment related. In consequence, the NOAEC were 0.964 mg/l for respiratory tract irritation and 2.783 mg/l for systemic toxicity.