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Repeated dose toxicity: inhalation

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sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11 April to 12 July 1990
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary study conducted according to GLP, and comparable to current guidelines.
Reason / purpose:
reference to other study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:
The study was comparable to OECD 413.
GLP compliance:
40 CFR, Part 792
Limit test:

Test material

Details on test material:
Two hydrogen fluoride (HF) cylinders were used, obtained from Matheson Gas Products. The first cylinder was numbered TA12299 and arrived on 07/11/89, the second cylinder was numbered TA12152 and arrived on 16/04/90. The material was a gas and was stored in the cylinder at room temperature. The purity, as given by the supplier, was 99.9%. The identity of the test material was confirmed at Battelle by Time-of-Flight mass spectrometry. An expiration period of 6 months from opening was provided. The first cylinder was opened on 19/01/90, and the second on 01/06/90.

Test animals

Fischer 344
Details on test animals and environmental conditions:
The animals were male and female Fischer 344 rats, obtained from Charles River Breeding Laboratories, Inc., NY. The rats were 5-6 weeks old on arrival. The rats were held in quarantine for 14 days, during which time they were observed twice daily for signs of disease or other abnormalities. Five animals of each sex were selected for serological analysis, and blood was collected from each animal during the quarantine period and at necropsy for titer determinations to common murine infectious agents. No significant titers were measured in any of the samples collected.
At the end of the quarantine period, the rats were weighed and randomly assigned to treatment groups by body weight, using Battelle's Xybion Path/Tox data capture system. The male body weights ranged from 129.8 to 167.2 g, and female body weights ranged from 93.8 to 107.7 g. Rats were identified with tail tattoos and cage tags.
Certified Purina Rodent Chow and water (City of Columbus Municipal Supply) were provided ad libitum, except during each exposure period where only water was provided. The rats were housed individually from arrival, in stainless steel wire mesh cages (Allentown Caging Company, NJ) on racks. One rack was used to house all animals assigned to a particular group. The temperature of the animal room was maintained at 67-77°F, and relative humidity was maintained at 40-70%. Fluorescent lighting was provided on a 12 hour light/dark cycle.

Administration / exposure

Route of administration:
Type of inhalation exposure:
whole body
other: nitrogen and air
Remarks on MMAD:
MMAD / GSD: The HF generated at the concenetrations used in this study remained in the vapour phase.
Details on inhalation exposure:
Test article atmospheres were generated from cylinders of pure hydrogen fluoride (HF) gas, and subsequently diluted with nitrogen and High Efficiency Particulate (HEPA)/Charcoal filtered room air to achieve the target concentrations. The flow of the gas from the cylinder was was directed through a stainless steel manifold containing three ruby orifice flow meters, from which the flow of gas was directed to individual chambers via separate stainless steel delivery lines. Flow of the gas through the orifices was controlled by the pressure on the cylinder. They cylinder was wrapped in a thermostatically controlled heating jacket to maintain control of the pressure in the cylinder. Nitrogen was then introduced, in varying amounts to dilute the HF gas. These gas mixtures were then directed to separate flow controlling devices. The finally dilution step was the addition of the HEPA/Charcoal filtered air, to achieve the target concentration at the inlet of the chamber. Airflow through the chamber inlets was driven by an exhaust blower system, maintaining a negative pressure (relative to the room) in the transport lines and exposure chambers. Special precautions were taken to minimise and control the high corrosivity and reactivity of the HF gas.
The rats were housed and exposed during the study period in Battelle-designed live-in exposure chambers (Model H1000, Hazleton Systems/Lab Products). Air flow through the chamber was diverted at the inlet to flow vertically down the inner surfaces of the chamber. The air was exhausted at the bottom centre chamber, below the lowest tier. The chambers contained a single column of cage batteries, comprised of three tiers. Each shelf holds a cage battering capable of housing 24 rats. Sample ports were located above each shelf, at both the front and rear.
The chamber environment was regulated to maintain chamber temperature at 72±5°F and relative humidity between 40 and 70%. Chamber parameters were measured at half hourly intervals. All chambers were operated at 13 to 17 air changes per hour (233 to 267 L/min), controlled by calibrated orifice plate flowmeters located on each chamber exhaust.
A pre-exposure validation of the system was conducted to ensure the system was operating as designed.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
A combination fluoride electrode (Model 96-09, Orion Research Inc) was used to measure the HF concentrations in sampled atmospheres from each exposure chamber, the room, and the post-scrubber exhaust. HF was trapped in a solvent-based impinger sampling device. Samples were collected at the following flow rates and durations: 10 ppm chamber the sample flowratw was 0.3 L/min for 5 min; in the 1 ppm chamber the flow rate was 1 L/min for 15 min, and for all other locations the flow rate was 3 L/min for 50 minutes. 25 mm Teflon filteres were used to check for the possibility of particulate aerosol; no significant particulate material was observed, therefore it was concluded that the HF remained in the vapour phase.
A least squares linear regression analysis was performed on the millivolt potential values versus the logarithm of standard concentration (0 - 1.2 ppm). The equation of the regression curve was used to calculate the chamber test material concentration.
Samples collected for HF electrode analysus were drawn from the mid level sampling port on the front of each inhalation chamber with a sampling probe that extended approximately 10 cm into the chamber. Samples for concentration determination were taken hourly during the exposure.
Exposure uniformity was evaluated by measuring the concentration of test article at 6 locations within each chamber; front and back of three exposure shelves. Total port variability, within port variability and between port variability were estimated.
Duration of treatment / exposure:
6 hr/day, 5 day/wk for 91 days (a total of 65 exposures)
Frequency of treatment:
5 days per week for 13 weeks
Doses / concentrations
Doses / Concentrations:
0, 0.1, 1.0 and 10.0 ppm
nominal conc.
No. of animals per sex per dose:
20 rats/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Rats were exposed for 6 hr/day, 5 day/wk for a total of 65 exposures. HF concentrations of 0 (filtered air alone), 0.1, 1.0 and 10.0 ppm were chosen, based on results obtained in the previous 14 day study (NOAEL = 1 ppm).
Rats were randomly assigned to treatment groups by body weight.
Positive control:
A positive control was not included.


Observations and examinations performed and frequency:
Observations for moribund or dead animals were made twice daily (am/pm) before and after each daily exposure, or at similar intervals on non-exposure days. All animals were examined weekly for clinical evidence of toxicity or other abnormalities.
Body weights were determined for each rat on Study Day -2 for randomisation, then on Day 1 prior to the first exposure, weekly thereafter, and prior to scheduled necropsy.
Sacrifice and pathology:
All animals that died prior to scheduled necropsy were necropsied within 16 hours of being found dead. Surviving rats were necropsied at the end of the exposure period. At necropsy, each animal was weighed, anaesthetised with sodium pentobarbital and killed by exsanguination. The following organs were weighed from animals that survived to scheduled necropsy: liver, kidneys (pair), testes or ovaries (pair), adrenals (pair), heart (excluding major vessels), spleen, brain, and lungs. Organ:body weight and organ:brain weight ratios were calculated.
The following tissues were grossly examined, dissected and preserved in 10% neutral-buffered formalin (eyes and tested were fixed in Bouin's solution): adrenals, bone (femur and marrow), brain, epididymis, oesophagus, eyes and optic nerve, gross lesions, harderian glands, heart and aorta, intestine, kidneys, larynx, liver, lungs with bronchi, mammary glands, mandibular lymph node, mesenteric lymph node, nasal cavity, thymic lymph node, ovaries, pancreas, parathyroid, ppharynx, pituitary, preputial or clitoral glands (paired), prostate, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin (dorsal midline), spinal cord, spleen, stomach, tail (for identification), testes, thymus, thyroid gland, trachea, tracheobronchial lymph nodes, urinary bladder, uterus, Zymbal's glands.

The respiratory tract, all gross lesions suspected to be exposure related, were embedded in paraffin, sectioned, stained with haematoxylin and eosin and submitted for light microscopy. In addition, all remaining preserved tissues from the air-only control and high-concentration HF (10 ppm) groups were embedded, sectioned, stained and examined microscopically. Target organs from the 0.1 and 1.0 ppm groups were embedded, sectioned, stained and examined microscopically.

Blood samples were collected from all rats prior to scheduled necropsy, by retro-orbital sinus puncture under sodium pentobarbital anaethesia. The following haematology paramters were measured on a Serono-Baker System 9000 Hematology Analyzer: haemoglobin, haematocrit, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, white blood cell count, reitculocyte count, morphological assessment of erythrocytes and platelets, white blood cell differential, absolute and relative.
Blood samples for serum chemistry were collected at the same time from the retro-orbital sinus.The following serum chemistry determinations were measured on a Hitachi 704 Analyzer: glucose, lactate dehydrogenase, alanine dehydrogenase, aspartate aminotransferase, alkaline phosphatase, total bile acids, urea nitrogen, creatinine, creatine kinase, total protein, albumin, globulin, cholesterol, bilirubin, calcium, chloride, phosphorous, sodium and potassium.
Other examinations:
Respiration rates were measured by visible counts on the first 10 rats per sex from each group. Measurements were made every day for the first 5 days of exposure, then weekly throughout the study.
Bartlett's test for homogeneity of variances, followed by a separate variance t-test of ANOVA as appropriate. Dunnett's test was used following ANOVA.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Five males and 1 female exposed to 10 ppm were found dead after Day 47: 1 died on Day 48, 2 died on Day 49, and 1 animal was found dead on each of the following days: 63, 78 and 79.

All clinical signs of toxicity were limited to animals in the 10 ppm group. The earliets clinical changes were a red ocular discharge occurring in both sexes on day 15. Roughened coat occurred in females on Day 15 and in males on Day 22. Other abnormalities including alopecia, thin appearance, hunched posture and nasal discharge occurred in both sexes after Day 20 and persisted until the end of the study. Two male rats developed polypnea on Day 50 continuing to Day 71. A wet urogenital region was observed in both sexes after Day 50 of the study. All animals in the lower concentration groups and in the control group were clinically normal throughout the study perod.

There were no differences in body weight gain between the controls and the 0.1 and 1.0 ppm groups. Group mean body weight values of male and female rats from the 10 ppm group increased only slightly during the study and at a decreased rate compared to controls. The 10 ppm male and female group mean body weight was significantly lower than controls from Day 8 until study termination. At week 13 the 10 ppm male group mean body weights were 21% lower than controls, whilst 10 ppm females were 6% lower than controls.

There were concentration-dependent minimal to mild increases in mean platelet counts from all groups of treated rats of both sexes. Mean platelet count differed from controls in a statistically significant manner in all HF-exposure groups of females and in the 10 ppm males. There were also minimal increases in group mean white blood cell counts of all HF-exposed groups, but the increase was only significant in the 10 ppm females. The slight increase in mean white cell counts of treated males were mainly due to increase in the numbers of segmented neutrophils, while in some groups of treated females there was also a contribution from increased lymphocyte counts. 10 ppm males and females had slight but statistically significant decrease in group mean erythrocyte counts. Group mean haematocrit and blood haemoglobin concentration were decreased in the 1 and 10 ppm rats. Mean corpuscular volume and mean corpuscular haemoglobin were significantly increased in 10 ppm males. Mean corpuscular volume was significantly increased in 10 ppm females.

Serum glucose concentrations were significantly decreased in all exposed females, and in 10 ppm males. Mean blood urea nitrogen was statistically singicantly increased in the 10 ppm females. Some other changes were seen (occasional decreased in enzymes, and increases in inorganic phosphorous and potassium concentrations) but there was no dose-response relationship so the changes were thought to have no toxicological significance.

There was a significant decrease in absolute kidney, testis, heart, spleen and liver weight values of 10 ppm males. There was a significant decrease in ovary weight in 10 ppm females. There was a significant increase in absolute brain and adrenal weight values in 0.1 ppm females. There was a significant decrease in absolute lung weight in 10 ppm males and females. There were significant increases in organ:body weight ratios for the adrenal gland, lung, heart and testis in the 10 ppm males. There was a significant increase in spleen:body weight ratios in 10 ppm females, and a decrease in ovary:body weight ratios in this group. Brain and kidney: body weight ratios were significantly increased in both male and female 10 ppm rats. Organ:brain weight ratios were significantly decreased in 10 ppm males for the kidneys, heart, spleen, lung and liver. Adrenal: brain weight ratios were significantly increased in 0.1 ppm females, and kidney:brain weight ratios were significantly increased in 10 ppm females. Ovary and lung to body weight ratios were significantly decreased in 10 ppm females.

All gross lesions observed at necropsy were typical of those observed in rats of this age and strain. Malocclusion was noted at the time of necropsy in 9 male and 2 female 10 ppm rats.

There were no lesions identified at histopathology that were considered to be exposure-related. The rats that died early revealed no lesions related to the cause of their deaths; they had a general shrunken appearance of parenchymal cells, and a stress-related lymphocyte depletion of lymphoid tissues. All of these changes were considered typical of those expected to occur in animals lacking adequate food or water over a period of time.

There were no apparent differences in respiratory rates between dose groups or sex in Weeks 1 through 7. Animals of both sexes in the three lower dose levels continued from Week 7 through Week 13 with no apparent changes. The 100 ppm animals displayed a marked decrease in respiration rates from Week 8 to 12, with an unexplained rise to average mean level at Week 13.

Effect levels

Dose descriptor:
Effect level:
0.88 ppm (analytical)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

On the first day of exposure the test article concentrations were below target, therefore the exposure period was extended for 1 hour.

The overall mean concentration values recorded for the 91 day exposure period were 0, 0.12, 0.88 and 9.21 ppm for the 0, 0.1, 1.0 and 10 ppm concentration levels, respectively.

Chamber uniformity measurements were completed during pre-validation and the first week of animal exposures, and found to be acceptable.

Applicant's summary and conclusion

The NOAEL can be considered to be 0.88 ppm (analytical), equivalent to 0.72 mg/m³.
Executive summary:

The potential toxicity of hydrogen fluoride (HF) gas was determined in Fischer 344 rats. Twenty rats per sex were exposed to HF gas at concentrations of 0 (filtered-air only), 0.1, 1.0 and 10 ppm, for 6 hours/day, 5 days/week for 13 weeks.

Six rats (5 males and 1 female) from the high dose group died during the study. There were marked body weight decreases in rats exposed to 10 ppm, accompanied by some decreases in selected absolute organ weight values. Clinical signs of toxicity were limited to the animals exposed to 10 ppm, and included red ocular discharge and rough coats. Some effects on haematology and clinical chemsitry parameters were detected, but the changes were slight and considered to be of minimal toxicological significance. The authors concluded that dental malocclusions noted at necropsy were likely to be a contributing factor to changes in blood values and body weights.

It was concluded that repeated exposure of rats to 10 ppm HF caused non-specific progressive toxicity, characterised by general malaise and loss of body weight. The authors also mentioned a decreased appetite and decreased food consumption, although there were no data reported to support this. The toxic effects of HF appeared to be more severe in males.

The NOAEL can be considered to be 0.88 ppm (analytical), equivalent to 0.72 mg/m³.