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Repeated dose toxicity: inhalation

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chronic toxicity: inhalation
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study; no restrictions, fully adequate for assessment

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes (incl. certificate)
Limit test:

Test material

Details on test material:
Purity: >99.97%

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Breeding Laboratories, Saint-Aubin-Les Elbeuf, France
- Age at study initiation: 47-50 days
- Housing: 4 to a cage (males) and 5 to a cage (females), in wire mesh stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

- Temperature (°C): 17-25
- Humidity (%): 41-92

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
other: unchanged (no vehicle)
Details on inhalation exposure:
The test material was led from cylinders via a pressure reducer, stainless steel tubing and four mass flow controllers to the mixing device of the inhalation chamber, where it was diluted with filtered air from the air conditioning system (filter efficiency: 80-90% for particles having a diameter of 0.2-0.4 µm) to obtain the desired concentration.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analyses of the test atmospheres were performed by a gas chromatograph type Intersmat GC 120 fitted with a flame ionization detector and used in a total hydrocarbon analyzer mode. The total hydrocarbon analyzer was connected with a microcomputer interfaced serial data acquisition system, which converted the output of the flame ionization detector from mV's into ppm's. Calibration of the total hydrocarbon analyzer was performed 7 times during the first year of exposure. During the second year (from 25 November) the FlO of the total hydrocarbon analyzer was checked for correct operation with a propane/air mixture at approximately weekly intervals. Consequently, the number of calibrations with vinylidene fluoride mixture could be reduced and was performed only 4 times during the second year of exposure.
The calibration was performed as follows:
A calibration gas was prepared by mixing known volumes of vinylidene fluoride with air. The detector response was calibrated at each of the four target concentrations. The calibration curve was drawn through three of the four calibration points and was described as a quadratic function;

C = a + bV + cV2

In which C = concentration in ppm's and V the response of the total hydrocarbon analyzer in mVs; a, band c are coefficients which can be calculated from the results of the calibration.
The remaining point (2500 ppm) deviated slightly from the calibration curve at most times. When calculating the concentration, the computer did not take into account the deviation of the 2500 ppm level, so that all values determined at this level had to be corrected for this deviation.
Samples of the test atmospheres were taken from one location in the inhalation chamber. A good distribution of vinylidene fluoride in the inhalation chamber has already been shown prior to the start of a previous inhalation study with vinylidene fluoride (TNO-CIVO Report no. V 85.449).
The nominal concentration could not be determined due to the fact that the batteries consisting of twelve cylinders each could not be weighed.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Doses / Concentrations:
150, 600, 2500, 10000 ppm
other: target concentrations
Doses / Concentrations:
149 (+/- 5), 597 (+/- 21), 2841 (+/- 86), 10011 (+/- 178) ppm
analytical conc.
No. of animals per sex per dose:
The control group consisted of a main group of 120 animals/sex and a satellite group of 20 animals/sex. The test groups consisted of a main group of 60 animals/sex and a satellite group of 20 animals/sex.
Control animals:
yes, concurrent no treatment
Details on study design:
After an acclimatization period of 20 days the study was started. Rats of the satellite groups were used to detect chronic adverse effects other than tumours, whereas rats of the main group were intended for carcinogenicity evaluation.
Positive control:


Observations and examinations performed and frequency:
- Time schedule: Twice a day

- Time schedule: Once a week (palpation for the presence of tumours and examined for clinical abnormalities)

- Time schedule for examinations: Prior to start of the study and once every week during the first 13 weeks, and once every four weeks thereafter

Food consumption was determined during the first 13 weeks of the study in all groups. Food consumption was measured per cage.



- Time schedule for examinations: Prior to start of the study and after 6 months
- Dose groups that were examined: All animals of the satellite groups (prior to start), 10 animals/sex of control- and high-concentration satellite groups (after 6 months)

- Time schedule for collection of blood: Week 13, 26, 52
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals of the satellite groups
- Parameters checked were differential white blood cell count, haemoglobin, mean corpusculair volume, mean corpusculair haemoglobin, mean corpusculair haemoglobin concentration, packed cell volume, red blood cells, reticulocytes, thrombocytes, white blood cells.

- Time schedule for collection of blood: Week 13 and 26 from the orbital plexus and from the abdominal aorta at autopsy in weeks 53-54
- Animals fasted: No data
- How many animals: All animals of the satellite groups
- Parameters checked were alanine amino-transferase (ALAT)/glutamic-pyruvic transaminase (GPT), albumin, alkaline phosphatase (ALP), aspartate amino transferase (ASAT)/glutamic-oxalacetic transaminase (GOT), bilirubin total, calcium, chloride, cholesterol, creatinine, creatine kinase (CK), γ-glutamyl transferase (γGT), glucose (blood), inorganic phosphate, ornithine carbamoyl transferase, potassium,, protein electrophoresis, sodium, total protein, triglycerides, urea

- Time schedule for collection of urine: Overnight in week 13, 26 and 52
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked were appearance, creatinine, density, γ-glutamyl transferase, pH, protein, glucose, occult blood, ketones, urobilinogen, bilirubin, potassium, sediment: erythrocytes, leucocytes, epithelial cells, amorph material, crystals, casts, bacteria, sperm cells and worm eggs, sodium, volume.


- Time schedule for evaluation and dose groups that were examined: In each inhalation chamber 10 additional animals per sex were placed for serological evaluation. In general, one male and one female rat were taken from each inhalation chamber and were sacrificed. Serological evaluation was carried out shortly after receipt of the animals (6/sex), at the start of exposure (6/sex), at week 22 (6/sex), at week 26 (6/sex), one year after exposure start (6/sex), 18 months after exposure start (5/sex), and two years after exposure start (4/sex).
Sacrifice and pathology:
Complete gross examination was done on all rats

The following organs were weighed of all rats of the satellite groups and of 10 male and 10 female survivors of each group at the end of the study: adrenals, brain, epididymides, heart, kidneys, liver, lungs with mediastinal lymph nodes, trachea and larynx, ovaries, pituitary, spleen, testes, thyroid.

See table 1
Other examinations:
Body weights were analysed by an analysis of co-variance (Cochran, W.G., 1957, Analysis of Covariance, Biometrics 8-57: 261-278) using preexposure (day 0) weights as the covariate. When group means were significantly different (p<0.05) individual pairwise comparisons were made using Dunnett's multiple comparison test (Dunnett, C.W., 1955, American Statistical Association Journal, 50: 1096-1121). Body weights and body weight gain were also analysed by one-way analysis of variance (ANOVA) (Bartlett's test, homogeneity of variances: Steel, R.G.D., J.H. Torrie, 1960, New York: McGraw-Hill., 347- 349) followed by the Dunnett's multiple comparison test.

Food intake was analysed by the analysis of variance followed by the Least Significant Difference tests (Steel, R.G.D., J.H. Torrie, 1960, Principles and Procedures of Statistics; with special reference to the biological sciences, New York: McGraw-Hill 106-107).

Analysis of variance followed by the Dunnett’s multiple comparison test were applied to the organ weights, organ-to-body weight ratios, haematological data (e.g. absolute cell counts), biochemical data, and urine analytical data (creatinine, y-GT, K, Na, volume and density).

Percentages of white blood cell counts, reticulocyte counts, globulines in blood, and urine pH were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney V-test (Siegel, S., 1956, Sciences, McGraw-Hill Kogakusha Ltd., 116-127). In case only the high-concentration group was compared with the control group, the Mann-Whitney V-test was performed.

Incidences of histopathological changes and numbers of deaths were analysed by the Fisher exact probability test. (Siegel, S., 1956, McGraw-Hill Kogakusha Ltd., 96-104).

All pairwise comparisons were two tailed. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
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):
not examined
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:
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:
At the end of the first year only a small number of males and females had died or were killed in extremis. During the second year mortality increased with the age of the animals. Exposure to viny1idene fluoride did not adversely affect the mortality incidences. Cantrol5 showed higher mortality incidences throughout the study than did rats exposed to 600 or 10,000 ppm. In males the differences occasionally reached a level of statistical significance.

A wide range of clinical signs were observed during the study scattered over the groups. The incidence of most of them was very low. Overgrowth of the incisors, a common finding in rats, occurred in all groups but at a higher incidence in males than in females. Other frequently observed signs were related to the orbital region or skin. Neither the kind of signs nor the frequency distribution over the groups could be related to treatment. Behaviour was similar in all groups.

Size, type and incidence of masses found during palpation did not reveal differences between groups which could be attributed to treatment. In females the major part of the masses was found in the mammary glands.

Generally, body weights were similar in all groups. However, when statistical analysis was performed (Co-variance followed by Dunnett's test) it appeared that rats of the 10,000 ppm group tended to gain less weight than controls during the first year of exposure. During the third half year mean body weights of the 10,000 ppm gr0up became similar to that of the controls. In addition, statistically significantly decreased body weights were observed in all treatment groups at several days. Most probably these differences could be explained by the lower mean body weight of control rats at day 0, compared to the mean body weights of rats of all the other groups. In case these data were analysed by analysis of variance (ANOVA) followed by Dunnett's test, generally no differences with the control group were observed except for increased body weights in all male test groups compared to control rats at day.
The decreased mean body weight of males of the 2500 ppm group at day 315 compared to day 287 was most probably due to a defect of the water system.
In all treatment groups, statistically significant increases and decreases in body weight gain compared to control rats were seen throughout the study period.
Therefore, the differences in body weight or body weight gain were felt to be isolated findings which could not be attributed to treatment.

Generally, food intake tended to be lower in the test groups than in controls, occasionally reaching statistically significant degrees. The differences with the controls, however, were only very slight. In addition the lower food consumption figures in rats exposed to 150, 600 or 2500 ppm were generally not accompanied by lower body weights.

Haemoglobin content (Hb) values were increased in all male test groups at day 90. At day 181 levels of Hb were still higher than those of the controls, but not to a statistically significant degree. The differences with the controls, however, were independent on the concentration. At day 356 haemoglobin content values were comparable in all groups. In addition, packed cell volumes were higher at day 90 and 181. At day 90 the differences with the controls were not concentration-related. In view of the differences in haemoglobin values between controls and males of the test groups at days 90 and 181, reticulocyte counts were determined in all male groups at these days. At day 356 reticulocytes were determined only in males of the 10,000 ppm group and of the control group. At day 90, reticulocytes were comparable in all male groups. At day 181, reticulocyte counts were higher in males exposed to vinylidene fluoride. The differences with the controls, however, did not reach a statistically significant level and they were not concentration-related. Reticulocytes were also determined in females of the highest concentration group and the control group at days 183 and 358; no differences were observed. Thrombocyte counts showed a wide variation between the individual animals especially after one year of exposure in male controls; a statistically significantly lower thrombocyte count was found only in males exposed to 2500 ppm. This lower count was considered an isolated finding unrelated to treatment.
At day 90 mean total white blood cell counts (WBC) in males showed a statistically significant decrease in groups exposed to 150 and 2500 ppm vinylidene fluoride. In the control group, however, there were two animals with very high values (27.8 and 30.4). If these two values were excluded from the calculation of the mean value, the mean value turned out to be 12.1, which value is completely comparable with that of the high concentration group. In view of the absence of any concentration-effect relationship the differences in total white blood cell count were considered casual findings unrelated to treatment. The statistically significantly decreased white blood cell count in males of the 150 ppm group at day 181 was an isolated finding not felt to be attributed to treatment.
The number of neutrophils was clearly higher in females exposed to 10,000 ppm vinylidene fluoride than in controls at day 92. This increase was caused by one animal (E 841) which had a value of 17.9 (x 109/l), whereas the other values in this group ranged from 0.8-3.0. Without this high value the mean number of neutrophils would have been 1.7, which is completely in line with that of the controls. The number of lymphocytes of this animal 8.9 (x 109/l) was in line with the mean value of 7.6 (x 109/l). The higher absolute number of neutrophils resulted in a significant increased percentage of neutrophils and a decreased percentage of lymphocytes. Since these changes were only observed at day 92, they were considered to be isolated findings unrelated to treatment.

In summary, the haematological changes observed showed inconsistency, a transient character and a lack of a clear concentration-response relationship, indicating that the differences observed are toxicologically irrelevant, although a relationship with treatment cannot be completely excluded.

At day 106 total protein (TP) was statistically significantly increased in females exposed to 2500 or 10,000 ppm vinylidene fluoride, and albumin was increased in females of the high concentration group in comparison with those of the controls, however, this increase in TP was not concentration related.
Apart from changes in total protein and albumin concentration, there were a few statistically significant differences in results of plasma analyses between test groups and controls. These changes scattered over the groups without showing any consistency and are considered isolated findings.

Urine pH values after one year of exposure in females were not determined in 2-hr urine samples but in 16-hr urine samples. This resulted in lower pH values. Urinary pH values showed a statistically significantly lower value in the male group exposed to 150 ppm vinylidene fluoride at day 190 and a higher value in females of the 2500 ppm group at day 367. Volume was decreased and density increased in males and females exposed to 600 ppm vinylidene fluoride for 3 months. The increases in urine of creatinine (Creat-U), γGT, potassium (K-U), and sodium (Na-U) in males and females exposed to 600 ppm vinylidene fluoride for 3 months seem to be directly related to the decreased volume.
None of the statistically significant differences in urine parameter values were consistent, most probably they are all casual findings.

Organs showing a clear morphological change (overt atrophy, or tumour) which might significantly have affected the weight of an organ were not included in the measurement. A few animals of the interim kill group died spontaneously before the end of the first year of the experimental period. Organ weights of these animals were not recorded.
Organ weights recorded after one year did not reveal changes which could be attributed to the test material.
All nominal organ weights of male rats exposed to 150 ppm vinylidene fluoride for 2 years were comparable with those of the controls or other groups. However, when expressed relative to body weights all organ weights of the 150 ppm group were lower than those of the controls. Brain, heart and epididymides weights reached statistically significant values. This phenomenon was not observed in females of this group. Four rats exposed to 10,000 ppm showed very high weight of the pituitary, resulting in an increased mean pituitary weight. At microscopy, these high weights appeared to be caused by the presence of tumours, which were not detected macroscopically.

Gross pathology of the satellite groups
Females of all test groups showed a relatively high incidence of enlarged pituitaries. However, this finding was not concentration-related. Ten out of 20 females of the low-concentration (150 ppm) group demonstrated either enlargement, or tumorous enlargement or tumorous nodules of the pituitary, whereas nine out of twenty females of the top-concentration group exhibited these changes. Moreover, in the low-concentration group four out of 20 females showed mammary masses, whereas in the top-concentration (10,000 ppm) group only one mammary mass was found. All macroscopical changes observed at day 370 represent normal background pathology of the Sprague Dawley rat and they were randomly distributed over test and control groups.

Gross pathology in rats of the main study (two-year)
Frequently occurring gross lesions in male and especially in female rats were tumorous changes of the pituitary. Pituitary tumours represented the main cause of death, of both male and female animals. In females a very high incidence of mammary tumours and masses was observed. The presence of such mammary masses often was the reason for euthanasia. Enlarged adrenals were also frequently observed in females.
In male animals tumorous skin lesions were frequently observed. Males also had a higher incidence of discoloured, granular and often enlarged kidneys, which is indicative of severe chronic nephropathy.
The remaining gross changes reported occurred in a few animals only, with no apparent preference for one of the groups.
The reported gross lesions represent normal background pathology of Sprague Dawley rats. All gross lesions were randomly distributed over test and control groups.

Microscopic examination in rats of the satellite groups
In the nose the olfactory epithelium in the dorsal meatus at the level of the second palatal ridge (level 4) showed signs of neuronal degeneration and basal cell hyperplasia in treated as well as control rats. In male rats the severity of olfactory epithelial degeneration tended to be slightly increased in the 10,000 ppm group. Basal cell hyperplasia in males showed a slight trend to increased severity in the 2500 and 10,000 ppm groups (P = 0.018 with Maxwell's trend test). In males, the total incidence of the nasal changes mentioned above appeared to be slightly, though not statistically significantly higher than in controls. In contrast, in females these types of changes did not show a concentration relationship. Therefore, the toxicological significance of these nasal findings remains questionable for the time being.
The other non-neoplastic - and neoplastic lesions occurred to about the same incidence and severity in control and test animals, or they occurred in a single animal or in a few animals only.

Microscopic examination in rats of the main group (two-year)
In male animals of the 10,000 ppm group the incidence of pulmonary alveolar (foamy) macrophages was slightly higher as compared with control rats. The pulmonary tissue showed focal accumulations of finely vacuolated macrophages, often accompanied by increased septal cellularity. The phenomenon always occurred to a very low degree, viz. a few small foci per animal. The presence of alveolar foamy macrophages in lung tissue is a common finding in rats, and belongs to the normal background pathology.
A large number of nasal changes was seen in test and control animals, and some of these changes showed statistically significant differences in incidence between test groups and controls. Differences of possible toxicological relevance are:
a) a decreased incidence of basal cell hyperplasia in males of the 150 and 2500 ppm groups,
b) an increased incidence of nest-like infolds (in both olfactory and respiratory epithelium) at 10,000 ppm in both sexes and at 2500 ppm in females,
c) increased incidences of olfactory epithelium degeneration in males of the 2500 and 10,000 ppm groups, and decreased incidences of this change in females of the 150 and 2500 ppm groups, and
d) increased incidences of olfactory epithelial cysts in males of the 600 ppm group, in males and females of the 2500 ppm group, and in females of the 10,000 pm group.
The other microscopic changes reported represent normal background pathology of Sprague Dawley rats, and were randomly distributed over test and control groups.
Type and incidence of neoplastic lesions were randomly distributed over test and control rats.
The tumour data did not reveal treatment-related shifts in benign or malignant tumour incidence, total number of tumours, or total number of tumour-bearing animals.

The minor nasal changes observed in (many) animals of both the satellite and the main groups, controls included are rather peculiar for rats indicating the changes not being primarily related to vinylidene fluoride. The higher or lower incidence of some of these changes in animals of the 600, 2500 and/or 10, 000 ppm groups as compared to cor, troIs might be ascribed to an enhancing or inhibiting effect of vinylidene fluoride on the development of these minor changes induced by an agent or condition to which all animals have been subjected. Under the (special nasal) conditions of the present study the "no-observed-adverse-effect-level" of vinylidene fluoride was found to be 150 ppm for males and 600 ppm for females. In view of
a) the nature and scantiness of the nasal changes observed, and
b) the fact that the adverse effects concerned higher incidences of changes also occurring in considerable (and unusual) numbers of controls,
the toxicological relevance of the nasal changes is considered negligibly small.

Serological investigations were negative at arrival of the animals but were positive for Sendai virus and PVM virus after 22, 26, 52, 78 weeks, and at the end of the study. In the course of the study increasing numbers of animals showed antibodies against Theiler (GD VII) and Kilham (KRV) virus but at the end of the study most animals were negative for Theiler and KRV antibodies. The positive reaction for MVM after 22, 26, and 78 weeks is most probably a cross reaction with KRV.

Effect levels

Dose descriptor:
Effect level:
10 000 ppm
Basis for effect level:
other: No treatment related effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion