Hydrogen fluoride

Endpoint summary

Description of key information

Comprehensive repeated dose oral toxicity data are available for sodium fluoride; read-across is therefore proposed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP study

Qualifier:

according to guideline

Guideline:

other: NTP protocol

Principles of method if other than guideline:

6-month study

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female rats were bred at the study laboratory. Breeder F344 rats (Harlan Industries, Indianapolis, IN) were placed on a low fluoride diet (<2.1 ppm fluoride) 1 month before monogamous pairing. Progeny that survived to weaning were distributed to weight classes and assigned to cages by a random number table. Rats were 5 to 6 weeks old when placed on study. Animals were houses five per cage with feed and water available ad libitum. Individual weights were recorded weekly throughout the studies. Water consumption was recorded daily by cage. The conditions the rats were kept in were; 22-24 degC, 40-60% humidity and 12 hours/day of fluorescent light.

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

Groups of ten rats of each sex were administered 0, 10, 30, 100 or 300 ppm sodium fluoride in deionized water, available ad libitum for 6 months.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analytical verification of doses. The concentrations are nominal.

Duration of treatment / exposure:

The study was 6 months in length.

Frequency of treatment:

The sodium fluoride in water was available ad libitum.

Remarks:

Doses / Concentrations:
0, 10, 30, 100 or 300 ppm
Basis:
nominal in water

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

other: see details of study design

Details on study design:

Groups of ten rats of each sex were administered 0, 10, 30, 100 or 300 ppm sodium fluoride in deionized water, available ad libitum for 6 months. All test animals receiving water supplemented with sodium fluoride were provided with a low fluoride (<2.1 ppm) semisynthetic diet throughout the study. The first two controls were only included in the female rat study.

Positive control:

Not applicable

Observations and examinations performed and frequency:

Rats were observed twice daily for mortality and morbidity, weighed initially, weekly and at termination. Clinical observations recorded daily. Food consumption recorded every other week for the first 13 weeks and for 1 week during each of the last 3 months. Water consumption was recorded daily.

Sacrifice and pathology:

Fluoride concentrations in bone, blood and urine were measured prior to necropsy. Necropsy was performed on all animals, with histopathological investigations at the two highest dose levels.

Other examinations:

No further observations

Statistics:

None reported

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

No deaths occurred. From Week 6, chalky-white teeth with an unusual wear pattern were observed in rats at the high dose level. During the latter stages of the study, teeth were trimmed due to their unusual length; chipping was also observed.

BODY WEIGHT AND WEIGHT GAIN

Bodyweights and food consumption were lower at 300 ppm in both sexes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

Water consumption was slightly reduced at 300 ppm.


GROSS PATHOLOGY

Thickening of the gastric mucosa at 100 and 300 ppm.

HISTOPATHOLOGY

The principal effects were observed on the incisor teeth (300 ppm males) and stomach (both sexes at 100 and 300 ppm). In 300 ppm males, degeneration of the enamel organ was apparent. Gastric effects were characterised by a diffuse hyperplasia of the glandular mucosa .


OTHER FINDINGS

Dose descriptor:

NOEL

Effect level:

30 ppm

Sex:

male

Basis for effect level:

other: Gastric pathology

Dose descriptor:

NOEL

Effect level:

30 ppm

Sex:

female

Basis for effect level:

other: Gastric pathology

Dose descriptor:

NOAEL

Effect level:

100 ppm

Sex:

male

Basis for effect level:

other: Reduced bodyweight, food and water consumption; dental fluorosis

Dose descriptor:

NOAEL

Effect level:

100 ppm

Sex:

female

Basis for effect level:

other: Reduced bodyweight, food and water consumption; dental fluorosis

Critical effects observed:

not specified

Dose (ppm)	Survival	Mean Body Weight			Final Weight relative to control (%)
		Initial	Final	Change
Male
Control	10/10	78 ±7	444 ±7	366 ±8	100
Control	10/10	78 ±7	450 ±7	372 ±10	101
Control	10/10	80 ±7	420 ±7*	339 ±8*	94
10	10/10	76 ±7	425 ±9	349 ±7	96
30	10/10	83 ±7	437 ±7	354 ±10	98
100	10/10	76 ±6	433 ±7	357 ±5	97
300	10/10	81 ±7	371 ±10**	290 ±8**	83
Female
Control	10/10	72 ±6	236 ±7	163 ±8	100
Control	10/10	67 ±6	234 ±4	167 ±6	99
10	10/10	75 ±7	232 ±3	156 ±6	98
30	10/10	69 ±7	234 ±6	166 ±7	99
100	10/10	69 ±7	235 ±4	166 ±8	100
300	10/10	70 ±7	212 ±3**	141 ±6	90

*Significantly different (P=0.05) from the control group by Dunn’s or Shirley’s test

**P<0.01

Conclusions:

There were no deaths throughout these studies. The only observed effects were signs of dental fluorosis and thickening of the mucosa and ulcer formation in the glandular stomach at 100 and 300 ppm.

Executive summary:

Sodium fluoride was shown to have an effect on the teeth and stomach of rats in this study. There was no mortality; bodyweights, food consumption and water consumption were reduced at the highest dose level of 300 ppm. Signs of dental fluorosis were apparent in all animals at 300 ppm and microscopically in males at 300 ppm. Local irritant effects on the gastric mucosa (hyperplasia and ulceration) were noted at 100 ppm and 300 ppm, however this local effect is considered likely to be a consequence of the method of administration and is not relevant to the human risk assessment.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

ANALOGUE APPROACH
Read-across from the source substance "Sodium fluoride" to the target substance "Hydrogen fluoride" based on structural similarity (RAAF - scenario 1 - analogue approach) whereby the source substance is used to predict the same property for the target substance to fulfil a REACH information endpoint.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

Available study data for sodium fluoride is being used for read-across to the target substance, hydrogen fluoride.

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Progeny of C57BL/N6 female and C3H/HeN male mice, obtained from Charles River Laboratories, Wilmington, DE, were used in this study. Mice were 4 to 6 weeks old when placed on study. Their diet consisted of a semisynthetic low fluoride diet. The mice were kept in groups of ten in cages set at a temperture of 22-24 degC and of relative humidity 40-60%. The fluorescent light was 12 hours per day.

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

Groups of 8-12 mice of each sex received 0, 10, 30, 100, 200, 300 or 600 ppm sodium fluoride in deionised water ad libitum for 6 months.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The concentrations are nominal concentrations.

Duration of treatment / exposure:

0, 10, 30, 100, 200, 300 or 600 ppm sodium fluoride in deionised water

Frequency of treatment:

Continous (ad libitum, in drinking water)

Remarks:

Doses / Concentrations:
0, 10, 30, 100, 200, 300 or 600 ppm
Basis:
nominal in water

No. of animals per sex per dose:

The method stated ten mice of each sex per dose group. However, some of the test animals were incorrectly sexed, resullting in 8-12 mice per sex per dose group.

Control animals:

other: One control group received de-ionised water and a low fluoride diet. One control group received sodium chloride and a low fluoride diet. An additional control group received standard diet.

Details on study design:

Groups of 8-12 mice of each sex received 0, 10, 30, 100, 200, 300 or 600 ppm sodium fluoride in deionised water ad libitum for 6 months. All test animals receiving water supplemented with sodium fluroide were provided with a low fluoride (<2.1 ppm) semisynthetic diet throughout the study. Three control groups were included in the studies of male and female mice; one received deionised drinking water and a low fluoride, semisynthetic diet, the second received sodium chloride supplemented deionized drinking water and a low fluoride, semisynthetic diet and the third received deionized water and a standard NIH-07 diet. At termination of the studies, the fluoride concentrations in urine, blood and bone were determined from samples collected from all surviving mice. Necropsy was performed on all animals, with histopathology at 300 and 600 ppm.

Positive control:

Not relevant

Observations and examinations performed and frequency:

Animals were observed twice daily for mortality and morbidity, weighed initially, once weekly and at termination. Clinical observations recorded daily. Food consumption recorded every other week for the first 13 weeks and for 1 week during each of the last 3 months. Water consumption was recorded daily.

Sacrifice and pathology:

Necropsy was performed on all animals, with histopathological investigation of animals at 300 and 600 ppm.

Other examinations:

Fluoride concentrations in bone, blood and urine measured prior to necropsy

Statistics:

Not reported.

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

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

Deaths occurred at 600 ppm (4 males, 9 females) and at 300 ppm (1 male). Signs of toxiicity (weakness, thin appearance, hunched posture) were seen at 600 ppm. Mice at 100, 200m 300 and 600 ppm had chalky white teeth; the lower incisors were more affected and were also chipped at higher dose levels.

BODY WEIGHT AND WEIGHT GAIN

Reduced weight gain was seen at 200, 300 and 600 ppm; food consumption was reduced in males at 600 ppm. Water consumption was unaffected by treatment.

GROSS PATHOLOGY

None

HISTOPATHOLOGY

Treatment-related findings were noted in the kidney, liver, testes and myocardium of decedents. Acute nephrosis was characterised by extensive multifocal degeneration and tubular necrosis and was diagnosed as the cause of death in these animals. Multifocal myocardial degeneration was also seen in two 600 ppm females. Liver changes consisted of scattered heptocellular hypertrophy and megalocytosis. The effects on the testes (degeneration/necrosis of the seminiferous tunules) were not considered to be directly related to treatment, but occur frequently in moribund mice. Effects were also noted on teh femur and (to a lesser extent) the tibia of mice at 50 ppm and greater. Changes are considered to be indicative of altered rates of bone deposition and remodelling. Effects on the teeth were seen at 300 and 600 ppm.


OTHER FINDINGS

The fluoride content of plasma, bone and urine increased with dose level.

Dose descriptor:

NOEL

Effect level:

< 50 ppm

Sex:

male

Basis for effect level:

other: Effects on bone

Dose descriptor:

LOEL

Effect level:

50 ppm

Sex:

male

Basis for effect level:

other: Effects on bone

Dose descriptor:

NOEL

Effect level:

50 ppm

Sex:

female

Basis for effect level:

other: Effects on bone

Critical effects observed:

not specified

Dose (ppm)	Survival	Mean Body Weight			Final Weight relative to control (%)
		Initial	Final	Change
Male
Control^a	9/9	16.9±0.4	40.2±1.0	23.3±1.1	100
Control^b	10/10	18.6±0.4*	41.6±0.6	23.0±0.7	103
Control^c	11/11	17.8±0.4	39.2±1.0	21.4±1.0	97
10	9/9	17.3±0.5	43.1±1.5	25.8±1.8	107
50	10/10	18.0±0.6	41.1±1.1	23.1±1.3	102
100	10/10	19.2±0.8	41.5±1.1	22.3±1.3	103
200	10/10	17.9±0.7	36.5±1.2	18.6±1.4*	91
300	7/8	18.8±0.7	38.1±1.1	19.0±1.4*	95
600	5/9	17.4±0.4	32.0±1.6**	14.8±1.9**	80
Female
Control^a	11/11	16.9±0.6	30.2±1.4	13.3±1.6	100
Control^b	10/10	18.6±0.4	31.5±1.0	12.9±1.1	104
Control^c	9/9	16.6±0.2	28.7±0.9	12.1±0.8	95
10	11/11	17.1±0.4	29.6±1.1	12.5±1.1	98
50	10/10	16.4±0.3	32.2±1.1	15.8±1.2	107
100	10/10	17.2±0.4	30.6±1.5	13.4±1.4	101
200	10/10	17.2±0.4	25.3±0.6**	8.1±0.7*	84
300	12/12	16.9±0.3	26.2±0.8*	9.3±0.7*	87
600	2/11	16.6±0.4	24.5±1.5	9.0±1.0	81

*Significantly different (P=0.05) from the control group by Dunn’s or Shirley’s test

**P<0.01

^a         Control group receiving semisynthetic, low fluoride diet and deionised water.

^b      Control group receiving semisynthetic, low fluoride diet and sodium chloride supplemented deionised water

^c      Control group receiving standard NIH-07 diet and deionised water.

Organs and Diagnoses	300 ppm	600 ppm
Male Animals initially in study Early deaths   Kidney Nephrosis, multifocal   Liver Megalocytosis, multifocal Syncytial alteration, multifocal   Myocardium Mineralization, multifocal   Testis Necrosis Tubule, degeneration, multifocal Tubule, multinucleated giant cells, multifocal   Female   Animals initially in study Early deaths   Kidney Nephrosis, multifocal   Liver Megalocytosis, multifocal Syncytial alteration, multifocal   Myocardium Degeneration, multifocal Mineralization, multifocal	8 1     1     1 1     1     1   1       12 0     0     0 0     0 0	9 4     2     4 4     4     3 2 1       11 9     2     7 7     2 4

Conclusions:

Skeletal effects of fluoride were seen at all dose levels in this study.

Executive summary:

In the 6 month studies in mice, 4/9 males and 9/11 females receiving 600 ppm sodium fluoride and 1/8 male given water containing 600 ppm died.

The fluoride content of urine and bone increased with the concentration of sodium fluoride in the drinking water in both sexes of mice. Bone fluoride concentration were as high as 14.8 µg/mg of ashed bone in male mice receiving 600 ppm sodium fluoride in water. The bone fluoride content found in mice was somewhat greater than that found in rats given comparable sodium fluoride content. This maybe due to a greater water intake on a body weight basis by mice than by rats resulting in higher exposures. Plasma fluoride concentrations in mice showed a good dose relationship and appeared increased in groups receiving water concentrations of 50 ppm of sodium fluoride or higher.

Histopathologic findings for mice are consistent with previously recognised toxic effects. The acute nephrosis observed in the kidneys was probably the most likely cause of death. Lesions were also observed on the incisor teeth, femur and tibia of mice

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Range-finding study for the NTP carcinogenicity study.

Qualifier:

according to guideline

Guideline:

other: NTP protocol

Principles of method if other than guideline:

14-day dose range-finding study in the rat.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female rats were obtained from Charles River Laboratories (Portage, MI) and were observed for 6 days. The rats were 5 weeks old when placed on the study. The rats were fed on a semisynthetic low fluoride diet, available ad libitum. Water was supplied in glass bottles with rubber stoppers and stainless steel sipper tubes that were chnaged every 3 days. The cages were kept at a temperature of 21-23 degC with a relavtive humidity of 40-60%. Fluorescent light was supplied for 12 hours per day.

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

0, 50, 100, 200, 400 or 800 ppm sodium fluoride in deionised water, available ad libitum.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Nominal concentrations.

Duration of treatment / exposure:

The study was 14 days in length. The sodium fluoride was available ad libitum in the drinking water.

Frequency of treatment:

The sodium fluoride was available ad libitum in the drinking water.

Remarks:

Doses / Concentrations:
0, 50, 100, 200, 400 or 800 ppm
Basis:
nominal in water

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes

Details on study design:

Groups of five rats of each sex received 0, 50, 100, 200, 400 or 800 ppm sodium fluoride in deionised water ad libitum. Animals were housed five per cage with semisynthetic low fluoride feed available ad libitum. Water consumption was recorded every 3 days. The rats were observed twice daily for signs of morbidity, mortality and signs of toxicity. They were weighed at the beginning, at the end of the first week and at necropsy. All animals were necropsied and tissues examined for gross lesions. No further investigations were performed.

Positive control:

Not relevant

Observations and examinations performed and frequency:

Water consumption was recorded every 3 days. The rats were observed twice daily for signs of morbidity, mortality and signs of toxicity. They were weighed at the beginning, at the end of the first week and at necropsy.

Sacrifice and pathology:

All animals were necropsied and tissues examined for gross lesions.

Other examinations:

No further examinations

Statistics:

Various

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):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

All male and female rats given water containing 800 ppm sodium fluoride died; 1/5 female given 400 ppm also died. No gross lesions were observed at necropsy. All groups of males and females surviving to the end of the studies gained weight except the group receiving 400 ppm. In this group, 4/5 male lost 5-31% of their initial body weight and 3/4 female lost 10-29% of their initial body weight.

The following signs of toxicity were noted in all animals in the two highest dose groups: dehydration and lethargy by day 4 and hunched posture by day 5. In addition, reduced water consumption recorded by cage for male and female in the second highest dose group (400 ppm).

Dose descriptor:

NOEL

Effect level:

200 ppm

Sex:

female

Basis for effect level:

other: Mortality, bodyweight effects, clinical signs, reduced water consumption

Dose descriptor:

NOEL

Effect level:

200 ppm

Sex:

male

Basis for effect level:

other: Bodyweight effects, clinical signs, reduced water consumption

Critical effects observed:

not specified

Dose (ppm)	Survival	Mean Body Weight			Final Weight relative to control (%)
		Initial	Final	Change
Male
Control	5/5	86 ±5	167 ±6	81 ±3	100
50	5/5	79 ±3	151 ±4	72 ±2	91
100	5/5	75 ±4	146 ±5*	71 ±1*	88
200	5/5	77 ±3	140 ±6**	62 ±4**	84
400	5/5	80 ±3	69 ±6**	-11 ±5**	41
800	0/5	79 ±2	-	-	-
Female
Control	5/5	80 ±4	133 ±2	53 ±3	100
50	5/5	74 ±3	128 ±3	53 ±4	96
100	5/5	79 ±4	128 ±2	49 ±2	96
200	5/5	77.0 ±5	128 ±4	51 ±3	96
400	4/5	80 ±4	79 ±14**	0 ±15**	59
800	0/5	77 ±3	-	-	-

*Significantly different (P=0.05) from the control group by Dunn’s or Shirley’s test

**P<0.01

Conclusions:

In the 14 day study, all male and female rats given water containing 800 ppm of sodium fluoride died. Effects at the second highest concentration (400 ppm) included weight loss, dehydration and lethargy. All other groups showed little signs of toxicity; males and females surviving to the end of the study gained weight.

Executive summary:

In the 14 day study, all male and female rats given water containing 800 ppm of sodium fluoride died; 1/5 female given 400 ppm died. No gross lesions were observed at necropsy. All groups, male and female, surviving to the end of the studies gained weight except the group receiving 400 ppm. In this group, 4/5 male lost form 5-31% of their initial body weight and 3/4 female lost from 10-29% of their initial body weight. The following signs of toxicity were noted in all animals in the two highest dose groups: dehydration and lethargy by day 4 and hunched posture by day 5. In addition, reduced water consumption was seen at 400 ppm.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP range-finding study.

Qualifier:

according to guideline

Guideline:

other: NTP protocol

Principles of method if other than guideline:

14-day range-finder for carcinogenicity study

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and Female B6C3F1 mice were obtained from Charles River Laboratories (Portage, MI) and were observed for 25 days prior to dosing. The mice were 5 weeks old when placed on study. Groups of five mice of each sex received 0, 50, 100, 200, 400 or 800 ppm sodium fluoride in deionised water ad libitum for 14 consecutive days. Animals were housed five per cage with semisynthetic low fluoride (12.7 to 14 ppm) feed available ad libitum. Water consumption was recorded every 4 days. The mice were observed twice daily for morbidity, mortality and signs of toxicity. They were weighed at the beginning of the studies and at the end of the first week and at necropsy. At the end of the study the mice were 7 weeks old.

The environmental conditions for the animals was 21-23 degC, 40-60% humidity and 12 hours/day of fluorescent light.

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

Groups of five mice of each sex received 0, 50, 100, 200, 400 or 800 ppm sodium fluoride in deionised water ad libitum for 14 consecutive days.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The concentrations are nominal

Duration of treatment / exposure:

14 days

Frequency of treatment:

The test substance in the drinking water was available ad libitum

Remarks:

Doses / Concentrations:
0, 50, 100, 200, 400 or 800 ppm
Basis:
nominal in water

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes

Details on study design:

Male and Female mice were obtained from Charles River Laboratories (Portage, MI) and were observed for 25 days. Groups of five rats of each sex received 0, 50, 100, 200, 400 or 800 ppm sodium fluoride in deionised water ad libitum for 14 consecutive days. The mice were observed twice daily for morbidity, mortality and signs of toxicity. They were weighed at the beginning of the studies and at the end of the first week and at necropsy.

Positive control:

Not relevant

Observations and examinations performed and frequency:

The mice were observed twice daily for morbidity, mortality and signs of toxicity. They were weighed at the beginning of the studies and at the end of the first week and at necropsy.

Sacrifice and pathology:

The mice were necropsied at the end of the study and any gross lesions were noted.

Other examinations:

No further examinations

Statistics:

Not applicable.

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

All mice survived to scheduled termination, except two high dose males that died on days 4 and 6.

Dose descriptor:

NOEL

Effect level:

400 ppm

Sex:

male

Basis for effect level:

other: Mortality, bodyweight effects, reduced water consumption, clinical signs

Dose descriptor:

NOEL

Effect level:

400 ppm

Sex:

female

Basis for effect level:

other: Bodyweight effects, reduced water consumption, clinical signs

Critical effects observed:

not specified

Dose (ppm)	Survival	Mean Body Weight			Final Weight relative to control (%)
		Initial	Final	Change
Male
Control	5/5	27.8 ±0.7	31.4 ±0.2	3.6 ±0.7	100
50	5/5	26.0 ±0.8	23.6 ±1.0**	-2.4 ±0.7*	75
100	5/5	28.4 ±0.2	25.6 ±0.5*	-2.8 ±0.5*	82
200	5/5	26.6 ±0.7	30.2 ±0.8	3.6 ±0.2	96
400	5/5	26.4 ±0.7	28.0 ±0.9	1.6 ±0.9	89
800	3/5	25.2 ±0.7*	20.0 ±2.5**	-6.0 ±2.1*	64
Female
Control	5/5	21.6 ±0.4	23.0 ±0.6	1.4 ±0.2	100
50	5/5	21.6 ±0.4	22.8 ±0.7	1.2 ±0.6	99
100	5/5	21.8 ±0.6	23.0 ±0.6*	1.2 ±0.2	100
200	5/5	21.0 ±0.3	23.0 ±0.5	2.0 ±0.3	100
400	5/5	21.2 ±0.2	21.6 ±0.4	0.4 ±0.4	94
800	5/5	21.6 ±0.2	19.6 ±0.5**	-2.0 ±0.3**	85

*Significantly different (P=0.05) from the control group by Dunn’s or Shirley’s test

**P<0.01

Conclusions:

All mice survived to scheduled termination, except two high dose males that died on days 4 and 6. The two high dose males that died began exhibiting signs of toxicity on day 4; they were noted to be thin, with stiff gait and hunched posture.

Executive summary:

All mice survived to scheduled termination, except two high dose males that died on days 4 and 6. The two high dose males that died began exhibiting signs of toxicity on day 4; they were noted to be thin, with stiff gait and hunched posture. Reduced water consumption was recorded for high dose males and females. Daily water consumption averaged approximately 30% for high-dose males and 60% for high-dose females in comparison with controls. Among the male mice, weight changes were variable, with the high-dose group having a significant decrease in body weight. Among female mice, weight losses occurred only in the high dose group.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 2, Non-GLP (or not specified) studies for 14-days or 6-months in male and female rats using Sodium Fluoride as a read-across surrogate analogue for Hydrogen Fluoride.

System:

other: Reduced bodyweight, food and water consumption, dental fluorosis.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 April to 12 July 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Proprietary study conducted according to GLP, and comparable to current guidelines.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

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:

yes

Remarks:

40 CFR, Part 792

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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.

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

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:

yes

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

Remarks:

Doses / Concentrations:
0, 0.1, 1.0 and 10.0 ppm
Basis:
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.

Statistics:

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.

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):

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.

Dose descriptor:

NOAEL

Effect level:

0.88 ppm (analytical)

Sex:

male/female

Critical effects observed:

not specified

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.

Conclusions:

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.72 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

Klimisch 1, OECD 413 GLP study in male and female rats for 6 hr/day, 5 day/wk for 91 days (total 65 exposures) (Placke et al, 1991).
Klimisch 1, OECD 412 GLP study in male and female rats for 6 hours/day, 5 days/week for 15 days (total 10 exposures) (Planke et al, 1990) using Hydrogen Fluoride.
Klimisch 2, Non-GLP, non-guideline study in female rats for 6 hours/day for 1-month (Sadilova et al, 1974) using Hydrogen Fluoride.

System:

other: Reduction in body weight

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 April to 12 July 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Proprietary study conducted according to GLP, and comparable to current guidelines.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

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:

yes

Remarks:

40 CFR, Part 792

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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.

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

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:

yes

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

Remarks:

Doses / Concentrations:
0, 0.1, 1.0 and 10.0 ppm
Basis:
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.

Statistics:

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.

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):

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.

Dose descriptor:

NOAEL

Effect level:

0.88 ppm (analytical)

Sex:

male/female

Critical effects observed:

not specified

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.

Conclusions:

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.72 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1, OECD 413 GLP study in male and female rats for 6 hr/day, 5 day/wk for 91 days (total 65 exposures) (Placke et al, 1991).
Klimisch 1, OECD 412 GLP study in male and female rats for 6 hours/day, 5 days/week for 15 days (total 10 exposures) (Planke et al, 1990) using Hydrogen Fluoride.
Klimisch 2, Non-GLP, non-guideline study in female rats for 6 hours/day for 1-month (Sadilova et al, 1974) using Hydrogen Fluoride.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose oral toxicity

 

No studies have been performed with HF, however comprehensive data are available for sodium fluoride. The repeated dose oral toxicity of HF and NaF are considered to be essentially identical, with the exception of likely irritant/corrosive effects of HF at high dose levels. The repeated dose oral toxicity of HF will be due to fluoride, therefore read-across from the comprehensive NTP dataset with the soluble salt NaF is appropriate.

 

In a 14 -day range-finding study with NaF in the rat, mortality was seen at drinking water concentrations of 400 and 800 ppm. Signs of toxicity (reduced weight gain, reduced water consumption, lethargy and dehydration) were noted in surviving animals in these groups. The NOAEL for this study was 200 ppm.

 

In a 14-day range-finding study in the mouse, mortality was seen at the highest dose level of 800 ppm; signs of toxicity (reduced weight gain, abnormal gait and posture, reduced water consumption) were also apparent at this dose level. A NOAEL of 400 ppm is determined for this study.

 

In a 6 -month rat study, the effects of exposure to NaF were limited to reduced weight gain, dental fluorosis, thickening and ulceration of the gastric mucosa at the highest dose level of 300 ppm; gastric effects were also seen at 100 ppm. The fluoride content of plasma, bone and teeth increased with dose levels. The NOEL for this study was 30 ppm, however these local effects are not considered to be relevant for the risk assessment therefore a NOAEL of 100 ppm can be determined.

 

In a 6 -month mouse study, mortality attributable to acute nephrosis was seen at the highest dose level of 600 ppm. Skeletal effects were seen in males at the lowest dose level of 50 ppm.

 

Repeated dose dermal toxicity

 

No studies are available. The effects of dermal exposure will be dominated by local irritation / corrosion. There is no evidence of significant dermal absorption of HF under exposure conditions where the integrity of the skin barrier is maintained. Testing for repeated dose dermal toxicity can therefore be waived on scientific grounds and for reasons of animal welfare. The effects of repeated inhalation exposure to HF have been adequately characterised; the effects of repeated exposure to fluoride are also well characterised.

 

Repeated exposure inhalation toxicity

 

In a published study (Sadilova et al, 1974), female rats were exposed to 1 mg/m3 HF 6 hours/day for 1 month. Effects were noted on the teeth, bones and respiratory tract. Two proprietary studies (Placke et al; 1990, 1991) give over-all NOAEL for repeated inhalatory exposure in male and female rats of 0.72 mg/m³(actual HF concentration) for a 6 hours per 5 days per week for 91 days exposure regimen. No adverse effects were noted at this concentration. At higher concentrations death, tissue irritation, dental malformations, haematological and biological changes and changes in several organ weights were observed.

 

Summary

 

Effects of repeated fluoride exposure in experimental animals were seen on the teeth, bones, respiratory tract and kidney. Evidence from epidemiological studies in humans also indicate that prolonged exposure to fluoride causes dental and skeletal effects.

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: other; other: bone

Justification for classification or non-classification

No classification is required for repeated dose toxicity.