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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
Only 30 animals/sex/concentration tested; body weights collected 2 times/month during the first 13 weeks; no food consumption data collected; deviations in haematology, clinical chemistry, urinalysis, and pathology in Tables 1-4 in Materials and Methods
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: >99.9%

Test animals

Species:
rat
Strain:
other: Crl:CD®Br
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 54 days
- Weight at study initiation: Mean male body weight week 0 was 217.6-245.6 g; mean female body weight week 0 was 159.3-166.2 g
- Fasting period before study: No
- Housing: Males and females of each concentration group were housed together and each of the four groups was housed in a separate animal room
- Diet (e.g. ad libitum): ad libitum, except during exposures
- Water (e.g. ad libitum): ad libitum, except during exposures
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±3°C
- Humidity (%): 50±10%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation exposure:
GGENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Four 4.6 m3 stainless steel and glass chambers, quadrangular in shape with pyramidal tops and bottoms
- Method of holding animals in test chamber: Not reported
- Source and rate of air: Not reported
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: The chambers were operated in a one-pass, flow-through mode. Atmospheres were generated by metering the test substance vapours from cylinders of liquid test substance, maintained at room temperature, through rotometers into the chamber air flow. The control chamber received dilution air only.
- Temperature, humidity, pressure in air chamber: 24±5°C, 50±10%, pressure not reported
- Air flow rate: approximately 1200 L/min.
- Air change rate: Not reported
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatograph equipped with a programmable recording terminal. Atmospheres were measured at approximately 30-minute intervals during each 6-hour exposure with daily average concentrations for each chamber calculated from these data.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Over the 106-week exposure period, the overall means of the weekly averages for the low, intermediate, and high concentrations were all 100% of design. While 93.4, 96.3, and 100% of the individual weekly average concentrations for the low, intermediate, and high exposures, respectively, were within 10% of design, all of the weekly averages for each exposure were within 15% of design. The overall means and standard deviations of the weekly average exposure concentrations were 0.2±0.001, 1.0±0.05, and 2.5±0.06%.
Duration of treatment / exposure:
Approximately 104 weeks
Frequency of treatment:
6 hours/day, 5 days/week, excluding holidays
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.2, 1.0, 2.5% (v/v)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.2±0.001%; 1.0±0.05%; 2.5±0.06%
Basis:
other: mean and standard deviation of the weekly average exposure concentration
No. of animals per sex per dose:
30 per sex per concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Male rats were exposed to 0 or 100000 ppm of the test substance in air for 6 hours per day, 5 days per week for 2 weeks, with half of the animals given a 14-day recovery period. During exposures, the rats appeared lethargic and were poorly responsive to sound. No other abnormalities attributable to the test substance were observed.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice monthly for the first 14 months, then once monthly for the remainder of the study
BODY WEIGHT: Yes
- Time schedule for examinations: Twice monthly for the first 14 months, then once monthly for the remainder of the study, and at sacrifice. One unscheduled weighing was added during the 12th month to monitor unusual weight loss observed in the preceding scheduled weighing interval.
FOOD CONSUMPTION: No
FOOD EFFICIENCY: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Approximately 1, 3, 6, 12, 18, and 24 months after start of the study
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: 10 rats/sex/concentration
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Approximately 1, 3, 6, 12, 18, and 24 months after start of the study
- Animals fasted: No data
- How many animals: 10 rats/sex/concentration
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Approximately 1, 3, 6, 12, 18, and 24 months after start of the study
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table No. 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 4)

HISTOPATHOLOGY: Yes (see table No. 4)

All high-exposure and control animals sacrificed at the 3- and 12-month interim and 24-month terminal examinations, and all animals from all groups which died or were sacrificed in extremis during the study, received histopathologic evaluation of the organs and tissues. Kidney and nasal tissues at the 3-month interim and 24-month final sacrifices, respectively, from all intermediate- and low-exposure rats received histopathological evaluation. All other organs and tissues collected at the 3- and 24-month sacrifices, and all organs and tissues collected at the 12-month sacrifice form intermediate and low- exposure rats, were processed to the block stage and held without histopathologic evaluation.
Statistics:
Clinical measurements were subjected to partially-nested and crossed analysis of variance. Organ weight and final body weight data were subjected to one-way analysis of variance and Dunnett’s test. The least significant differences from control values were calculated whenever the ratio of variances (F-ratio) indicated that differences existed among the study groups. Body weight data were subjected to one-way analysis of variance. Significance was judged at p≤0.05 level of significance. The results of histopathologic examination were analyzed by the Fisher’s Exact test for differences between control and exposed groups and by the Mantel-Haenszel test for a dose-related trend.

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: Compared to controls, the incidences of ocular/nasal discharges and wet/stained perinea among male and female rats at 2.5% and of stained body/face among female rats at 2.5% were significantly elevated with dose-response trends apparent for the latter 2 signs. In addition, the incidences of swollen ears were significantly elevated in males at 0.2% and in males and females at 2.5%, with a dose-related trend in the females. Other differences in the incidences of clinical signs, which were less clearly related to exposure, consisted of increased and decreased occurrences of alopecia and masses, respectively, in males at 0.2%. Largely as a reflection of the changes in clinical signs noted above, the incidences of all signs combined were significantly elevated with respect to controls in males and females at 2.5% with dose-related trends among the groups in both sexes. While all male groups and females at 0.2% exhibited slightly-earlier incidences of and greater total mortality than the control groups, these differences were not statistically significant and were considered neither to be related to exposure nor to be biologically significant. In addition, no differences were noted in probable or contributing causes of death or morbidity between control and test groups.

HAEMATOLOGY: Male rats in the 1.0 and 2.5% groups exhibited elevated hematocrit and mean corpuscular volumes, but decreased mean corpuscular haemoglobin concentrations during the study. The 0.2% male group showed slightly-reduced erythrocyte counts throughout the first 12 months, but not when evaluated over the entire study period. Throughout the study, female rats exhibited increased mean corpuscular volumes at 1.0 and 2.5%, increased mean corpuscular haemoglobin at 1.0%, decreased mean corpuscular haemoglobin concentration at 1.0 and 2.5%, and decreased hematocrit at 0.2%. All test substance exposed male and female rats presented decreased relative numbers of monocytes and eosinophils, respectively, throughout the study. Also throughout the study, the 0.2% female group exhibited decreased relative numbers of neutrophils and increased relative numbers of lymphocytes and monocytes. Increased relative numbers of lymphocytes and leukocytes were observed in females at 0.2 and 1.0%, respectively, during the first half, but not over the entire study.

CLINICAL CHEMISTRY: Serum creatinine was consistently elevated with respect to controls in females at 1.0 and 2.5%. Creatinine was also elevated in males at 2.5% after the first month, but was comparable to controls for the study as a whole, while it was decreased in males at 0.2% over the duration of the study. Serum bilirubin was elevated throughout the study in all treated female groups. Blood urea nitrogen was decreased in males at 0.2 and 2.5% during the first 12 months and in all treated males groups when evaluated for the whole study period. Glutamic-pyruvic transaminase activities of males at 1.0% were higher than control values over the duration of the study. Total protein was reduced in males at 0.2% over the duration of the study, while it was reduced in females at 0.2 and 2.5% only during the first 12 months

URINALYSIS: A significant and dose-dependent increase in urinary fluoride concentration occurred in males and females at 1.0 and 2.5% at all evaluation periods and in males at 0.2% when evaluated over the entire study. Likewise, a significant and dose-dependent increase in the amount of fluoride excreted also occurred in females at 1.0 and 2.5 % and in all treated male groups at all evaluation periods. Urinary volume and osmolality were increased and decreased, respectively, in females at 2.5% over the duration of the study. Males at 2.5% exhibited increased urobilinogen with respect to controls when evaluated over the entire study period.

ORGAN WEIGHTS:
3-Month Sacrifice: Male and females at 2.5% exhibited a significantly lower relative kidney weight when compared to controls. The relative kidney weight of males at 0.2 and 1.0% were slightly, but not significantly, lower than controls suggesting a dose-response kidney weight decrease among the treated male groups. A number of other differences in mean absolute and relative organ weights were observed between treated and control groups, which included: increased absolute but not relative heart, stomach, and adrenal weights in males as 0.2%; decreased absolute liver and spleen weight in males at 2.5% and decreased relative liver and spleen weights in males at 1.0 and 2.5%; decreased absolute and relative thymus weights in males at 2.5%; decreased relative lung weights in males at 0.2 and 2.5%; decreased relative testes weights in males at 1.0%; decreased relative pituitary weights in males at 0.2%; increased absolute but not relative stomach weights in females at 0.2% and adrenal weights in females at 0.2 and 1.0%; decreased absolute brain weight in females at 2.5% and relative brain weight in females at 0.2 and 2.5%; decreased absolute heart weight in females at 2.5% and relative heart weights in all treated female groups; decreased absolute lung weight in females at 1.0 and 2.5% and decreased relative lung weights in all treated female groups; decreased absolute spleen weights in females at 2.5% and decreased relative spleen weights in all treated female groups; increased absolute pituitary weights in all treated females and increased relative pituitary weights in females at 1.0 and 2.5%; and decreased relative liver weights in females at 2.5%.

12-Month Sacrifice: Increased absolute but not relative lung weights in males at 1.0%, decreased absolute thymus weights in males at 0.2% and decreased relative thymus weights in males at 0.2 and 1.0%, increased relative heart and stomach weights in males at 2.5%, decreased absolute pituitary weights in females at 0.2 and 1.0% and decreased relative pituitary weights in all treated females, and decreased relative liver weights in females at 2.5%.

24-Month Sacrifice: Increased absolute and relative lung weights in females at 0.2 and 1.0%, increased absolute stomach weight in females at 2.5% and increased relative stomach weight in all treated female groups, increased relative heart weight in females at 0.2%, and increased relative liver weight in females at 2.5%

HISTOPATHOLOGY:
3-Month Sacrifice: Renal tubular damage consisting of slight cytoplasmic vacuolation, luminal dilatation and the presence of occasional vesiculated nuclei were observed in 4 of 10 male and 7 of 10 female rats at 2.5%. Similar lesions were not observed in rats from either of the test substance exposure groups. Generally, histopathologic changes documented by the peer review were in close agreement with the original interpretations. There was no distinct evidence of test substance-induced toxicity or carcinogenicity in any tissue examined as part of the review. Preparation of additional kidney sections indicated that renal tubular changes recorded by the original pathologist in female rats sacrificed after 3 months of exposure were the result of tissue processing artifact rather than treatment-related nephrotoxicity.

24-Month Sacrifice: Atrophy of the nasal olfactory epithelium was observed in animals from all but the females at 1.0% and all controls. The mucosal atrophy was equally severe in all treatment groups affected. A similar lesion, only focal in nature, and described as focal mucosal metaplasia, was seen in all treated and control animals. The incidences of focal mucosal metaplasia in all of the treated groups were clearly not dose-related. The incidence of mucosal atrophy was statistically significant in males and females at 0.2 and 2.5%, although an analysis for dose-relate trend was significant only for the males. One male and 1 female rat at 2.5% each revealed an adenoma which was located in the respiratory epithelial lining of the nasoturbinate. The incidence of the tumour was not statistically significant in either group when compared with controls. Three males at 2.5% revealed osteomas that appeared to originate from the skull and replaced the olfactory and frontal regions of the bran in each rat. The incidence of osteoma in this group was not statistically significant when compared to the male controls. Generally, histopathologic changes documented by the peer review were in close agreement with the original interpretations. There was no distinct evidence of test substance-induced toxicity or carcinogenicity in any tissue examined as part of the review. Atrophy of the olfactory epithelium was confirmed by the peer review to be present in small numbers of females exposed for 2 years. These nasal changes, however, usually were unilateral in distribution and were entirely consistent with olfactory lesions which recently have been described as spontaneous alterations in aging rats. Further, group incidence trends for olfactory atrophy were inconsistent with exposure concentration gradients. Olfactory changes, therefore, were not regarded as treatment-related.

Effect levels

Dose descriptor:
NOAEC
Effect level:
2.5 other: %
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Urine Fluoride Concentrations

Months on Test (males)

1

3

6

12

18

24

Concentration (%)

Urine Fluoride (ppm)

0

1.7

1.4

3.7

3.4

10.9

2.5

0.2

2.9

1.9

6.9

7.1

38.9

4.2

1.0

6.5

5.6

13.7

16.4

44.8

8.5

2.5

8.1

11.8

19.2

30.9

88.4

13.0

Months on Test (females):

3

6

12

18

24

Concentration (%)

Urine Fluoride (ppm)

0

4.2

2.3

3.5

2.8

10.8

2.2

0.2

5.3

2.4

5.3

4.5

17.0

4.1

1.0

9.4

4.9

11.8

12.5

32.0

13.5

2.5

9.6

5.3

14.2

14.6

42.8

10.5

 

Female Organ Weights (24-Month Sacrifice)

Female Exposure

0%

0.2%

1.0%

2.5%

 Absolute lung weight (g)

1.9939

2.1244

2.1166

2.0562

 Relative lung weight (g)

0.3870

0.4447

0.4415

0.4115

 Absolute stomach weight (g)

2.5302

2.6428

2.6200

2.7792

 Relative stomach weight (g)

0.4857

0.5494

0.5446

0.5448

 Relative heart weight (g)

0.2570

0.3176

0.2966

0.2917

 Relative liver weight (g)

3.3324

3.4723

3.6701

3.8151

 

Histological Effects of Nasal Olfactory Epithelium (24-Month Sacrifice)

Exposure

0%

0.2%

1.0%

2.5%

 Males

 No. of nasal tissues examined

97

87

92

89

 Focal mucosal metaplasia

57

21

21

21

 Mucosal atrophy

0

5

2

8

 

 Females

 No. of nasal tissues examined

95

88

91

95

 Focal mucosal metaplasia

22

17

6

23

 Mucosal atrophy

0

9

0

17

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

NOAEL = 2.5%
NOEL = Not determined
LOEL = 0.2%
Executive summary:

Male and female rats were exposed, whole body, via inhalation for 6 hours a day, 5 days a week to 0, 0.2, 1.0, or 2.5% (v/v) of the test substance for 2 years. Ten rats per sex per concentration were subjected to clinical pathological evaluation after 1, 3, 6, 12, 18, and 24 months on test. An additional 10 rats per sex per concentration at 3 and 12 months on study and all surviving rats at 24 months on study were sacrificed and, together with animals found dead or sacrificed in extremis, were subjected to gross pathological examination.  All high-concentration and control animals plus all animals found dead or sacrificed in extremis were subjected to complete histopathological evaluation while only the kidneys and nasal tissues at the 3-month interim and 24-month final sacrifices, respectively, from the low- and intermediate-concentration animals were examined histopathologically. 

The overall means and standard deviations of the weekly average exposure concentrations were 0.2±0.001, 1.0±0.05, and 2.5±0.06%. Male and female rats at 2.5% displayed increased incidences of swollen ears, ocular/nasal discharges and wet/stained perinea while the females at 2.5% also exhibited an increased incidence of staining of the body and face. A significant and dose-related increase in urinary fluoride concentration and excretion was observed in males and females at 1.0 and 2.5% at all evaluation periods and in males at 0.2% when evaluated over the entire study. Elevated serum creatinine in females at 1.0 and 2.5% and increased and decreased urinary volume and osmolality, respectively, in females at 2.5% over the duration of the study, together with depressed relative kidney weights and renal tubular alterations in all rats at 2.5% at 3 months, suggest a mild, reversible alteration in the renal structure of rats at 2.5% with persistent slight interference with renal function in females at 2.5%. A test substance-induced atrophy of the nasal olfactory epithelium was noted only at final sacrifice in some rats from all but the females at 1.0%. Although a statistically-significant trend for incidence of the lesion was noted in males, but not females, there was no dose-response relationship in terms of severity or the time course to development of the lesion. In addition, a high incidence of a similar lesion, only more focal in nature, was observed in all treatment and control groups. A nasal adenoma in one male and one female at 2.5% and an osteoma originating from the skull in 3 males at 2.5% were observed at the final sacrifice. The incidence of neither tumour was statistically significant and each was considered to be of unclear biological significance. In conclusion, the test substance administered by inhalation intermittently over a 2-year period produced a significant and dose-related increase in urinary fluoride concentration and excretion; some adverse clinical signs in rats at 2.5%; a low incidence of atrophy of the nasal olfactory epithelium at all test concentrations in males and in females at 0.2 and 2.5%; and a mild, reversible alteration in the renal structure of rats at 2.5% with persistent, slight interference with renal function in females at 2.5%.  Generally, histopathologic changes documented by the peer review were in close agreement with the original interpretations. There was no distinct evidence of test substance-induced toxicity or carcinogenicity in any tissue examined as part of the review. Preparation of additional kidney sections indicated that renal tubular changes recorded by the original pathologist in female rats sacrificed after 3 months of exposure were the result of tissue processing artifact rather than treatment-related nephrotoxicity. Atrophy of the olfactory epithelium was confirmed by the peer review to be present in small numbers of females exposed for 2 years. These nasal changes, however, usually were unilateral in distribution and were entirely consistent with olfactory lesions which recently have been described as spontaneous alterations in aging rats. Further, group incidence trends for olfactory atrophy were inconsistent with exposure concentration gradients. Olfactory changes, therefore, were not regarded as treatment-related. The peer review indicated that the original pathology methods and interpretations were adequate in scope and accuracy, and that the test substance is not toxic or carcinogenic to CD rats under the conditions of the study.