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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.12.1999 to 09.06.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.4300
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc.
- Age at study initiation: Six weeks
- Weight at study initiation: Males: 92-130 g; Females: 80-112 g.
- Fasting period before study: No, but animals not fed during exposure
- Housing: Groups of up to five animals of same sex in Makrolon type IV cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Ten days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3
- Humidity (%): 40-70
- Air changes (per hr):10-15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14.12.1999 To: 09.06.2005

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: filtered air
Remarks on MMAD:
MMAD / GSD: The saturated vapour concentration of D5 was experimentally evaluated to be 180 ppm, and therefore is expected to be in vapour phase without liquid or mist particles at the concentration of 160 ppm.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Sealed chambers used for group isolation. Constructed from stainless steel, with glass doors. The chamber contained nine stainless steel wire cage units with excreta pan below each cage unit.
- Method of holding animals in test chamber: wire cage
- Source and rate of air: Compressed air (40 l/min) was supplied into the glass flasks and allowed the liquid to equilibrate with the temperature of the warm walls of the container. The vapour produced then passed through a pipe and was then mixed and diluted with 380 l/min of the filtered air to the chamber inlet duct. It passed through an ULTRA filter JK-S-19/30 filter before entering the exposure chamber. The temperature of the D5 vapour in the round flask was approx. 30oC in Group 2 (10 ppm), 50oC in Group 3 (40 ppm) and 80oC in Group 4 (160 ppm).


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration in each chamber of the dose groups was determined daily, approximately five times per hour of exposure. Concentrations were determined by GC analysis.
Duration of treatment / exposure:
Sub-group A: interim sacrifice after 6 months; 26 weeks.
Sub-group B: interim sacrifice after 1 year; 52 weeks.
Sub-group C: sacrifice after 2 years, 1 year of exposure and 1 year of recovery.
Sub-group D: oncogenicity phase; up to 106 weeks.
Frequency of treatment:
Daily, 6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0.15 mg/L air (nominal)
Remarks:
10 ppm (nominal)
Dose / conc.:
0.6 mg/L air (nominal)
Remarks:
40 ppm (nominal)
Dose / conc.:
2.42 mg/L air (nominal)
Remarks:
160 ppm (nominal)
No. of animals per sex per dose:
Subgroup A: 6; subgroup B: 10; subgroup C: 20; subgroup D: 60.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest exposure concentration is the highest vapour concentration that can be repetitively and reliably generated in whole-body exposure chambers for a study of this duration.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: to investigate reversibility of adverse effects
- Post-exposure recovery period in satellite groups: one year
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, as soon as possible following each exposure.
- Cage side observations for clinical signs of toxicity: behaviour, body position, respiration, nasal and ocular changes.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice during acclimitisation, weekly during weeks 2 to 14, then once every two weeks thereafter until the end of the study.


BODY WEIGHT: Yes
- Time schedule for examinations: During acclimitisation on day 1, 6 and on the day before the first exposure. The once weekly for the first 14 weeks. Thereafter the animals were weighed every four weeks until termination. In sub-groups C and D animals were weighed at the start of the week of terminal sacrifice. Animals were always weighed prior to daily exposure.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: All animals in sub-group B, C and D before the start of exposure, and all surviving animals in control and highest dose groups of sub-group B, the last 10 animals/sex of sub-group C, and the first 10 animals/sex of sub-group D in control and highest dose groups prior to necropsy.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, and 12 months of exposure
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: First ten animals/sex/group from sub-group C
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3, 6, and 12 months of exposure
- Animals fasted: Yes
- How many animals: First ten animals/sex/group from sub-group C
- Parameters checked in table 1 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: 3, 6, and 12 months of exposure
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table were examined.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

All animals that survived until sacrifice as well as all moribund animals were sacrificed. All macroscopic abnormalities were described and reported. All gross masses and clinically observed growths were confirmed at necropsy.

Subgroup A (6 month sacrifice): A portion of the liver was collected from all animals. A partial necropsy was performed on all animals in this group. Blood from cardiac puncture, peri-renal fat, abdominal fat and brown fat were also collected for determination of D5 concentration.
Subgroup B (sacrificed after one year): A portion of the liver was collected from all animals. Organ to body weight and organ to brain weight ratios were calculated. A complete necropsy was performed on all animals that died, were euthanised moribund or sacrificed at scheduled necropsy.
Subgroup C and D (sacrificed after 24 months): Organ to body weight and organ to brain weight ratios were calculated. A complete necropsy was performed on all animals that died, were euthanised moribund or sacrificed at scheduled necropsy.

Microscopic examinations were performed on control and highest dose group from sub-groups B, C and D. The lungs, liver, kidneys, nasal cavities and gross lesions and tissue masses were examined from all animals in groups 1, 2, 3 and 4 of sub-groups B, C and D.
Other examinations:
None
Statistics:
Analysis was two-tailed for significance levels of 5% and 1%. Generally, means are presented with standard deviations. Analysis of body weight, as well as organ weights and clinical pathology were analysed by a one way analysis of variance followed by comparison of control group to each treated group y Dunnett's test. The Steel test was applied instead of Dunnett's test if the data could not be assumed to follow a normal distribution. Fisher's Exact test was used to test for statistical significances between groups for the macroscopic and ophthalmoscopic data. Statistical analysis of histopathology data were recorded.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: No treatment-related effect on mortality. Approximately one third of animals died in sub-groups C and D. There were no clinical signs that were considered to be related to the exposure to D5.

BODY WEIGHT AND WEIGHT GAIN: No statistically significant treatment-related differences between D5 treated groups and controls.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related adverse effects.

There were no major effects of toxicological significance on haematology, clinical biochemistry and urinalysis data. However, for some parameters minor intergroup differences were recorded between treated rats and controls at different time points of the treatment.

HAEMATOLOGY: The only possible effect on haematology parameters was confined to reduction in the red blood cell count with associated changes in the mean cell volume and mean cell haemoglobin values in males at 40 and 160 ppm. However, the change was temporary and minimal in degree and therefore considered to be of no toxicological importance.

CLINICAL CHEMISTRY: The decrease in urea concentration and increase in cholesterol/triglycerides, proteins and gamma glutamyl transferase in the females were possibly related to treatment with D5 and suggest metabolic adaptive changes, primarily related to the liver. The only other change considered to be related to treatment was an increased calcium level in males and females at 160 ppm. No toxicological relevance is associated with these findings since the changes were minimal in degree and unaccompanied by any pathological findings.

URINALYSIS: Urinalysis changes (especially those in males after three months) were small, not present at subsequent measurements, had no pathological correlates and therefore were considered to be of no toxicological importance.

ORGAN WEIGHTS: The only organ weight changes that were considered to be possibly related to treatment with D5 were the increased liver weights in females after 6 and 12 months and in males after 2 years. However, this finding was not present in males after 6 and 12 months or females after 2 years, showed no relationship to dose (not apparent in females at 40 ppm) and there were no correlated findings at histopathological examination. Therefore this finding could be the consequence of a transient metabolic adaptation without any toxicological relevance.

GROSS PATHOLOGY: There were no treatment-related masses found in any group. See also Section 7.7.

HISTOPATHOLOGY: NON-NEOPLASTIC: The statistically significantly increased incidence of hyaline inclusions in the nasal respiratory/olfactory epithelium was noted in male and/or female rats of group 4 (160 ppm) sacrificed after 6, 12 and 24 months and are considered to represent a non-specific exposure-related effect. An increased incidence of hyaline inclusions was noted also in high dose males after the recovery period. It was not clear from this study whether or not this increase was related to the exposure. Histomorphologic changes in the nasal cavity were consistent with chronic inhalation of some mildly irritant chemicals but are also commonly observed in ageing rats. Since there were no other changes associated with a response to an irritant, such as an inflammatory cell infiltration or degenerative changes to the epithelium, the finding was considered to be non-specific and of low toxicological importance.

HISTOPATHOLOGY: NEOPLASTIC: See Section 7.7.

OTHER: TISSUE CONCENTRATIONS: Determination of the D5 levels in plasma, fat and liver following six months of exposures showed an increase with the dose, with slightly higher values in the fat and liver recorded for females compared with males.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
General toxicity
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No toxicologically relevant adverse effects at any concentration tested. Excluding local effects in nasal cavity.
Dose descriptor:
NOAEC
Remarks:
Carcinogenicity
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on uterine tumours following 24 months exposure to 160 ppm being not relevant to humans.
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
40 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on respiratory tract irritation

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a two-year inhalation combined chronic toxicity and carcinogenicity study in rats conducted to an EPA guideline and to GLP (reliability score 1) the NOAEC for general toxicity was ≥160 ppm (2.42 mg/l; the highest dose tested). Local effects on the nasal cavity and adaptive increases in liver weights (with no microscopic findings) in females were observed at 160 ppm. The NOAEC for carcinogenic effects was 40 ppm (0.6 mg/l) based on uterine tumours at 160 ppm.