Perchloric acid

Endpoint summary

Description of key information

Perchloric acid repeated toxicity evaluated by read-across with ammonium perchlorate.
Reversible thyroid hypertrophy/hyperplasia observed at 10 mg/kg/day dose on rat treated orally for 90 days.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Sufficiently compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions. Justification for read-across : Perchloric acid, once absorbed in the general circulation is expected to be transformed into perchlorate moiety because of the blood buffering effect. Mammalian toxicity data of a perchlorate salt : ammonium perchlorate (CAS no. 7790-98-9), has been used for read across where data gaps for perchloric acid exist (repeated toxicity).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Highest dose chosen with the aim to induce antithyroid effects, but not based on dose-limiting toxicity. No histological examination of Peyer’s patches.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, NY.
- Age at study initiation: approximately 5 weeks of age
- Weight at study initiation (day -1): 195-197 g (M), 151-153 g (F)
- Fasting period before study: no
- Housing: individual suspended stainless steel wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (PMI Rodent Meal #5002, Purina Mills, Inc.), ad libitum
- Water (e.g. ad libitum): reverse osmosis water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2° C
- Humidity (%): 50 ± 15%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

VEHICLE
- Nature: reverse osmosis drinking water
- Justification for use and choice of vehicle: high solubility of the test item in water
- Analyzed and found to contain no detectable nitrate, a potential interference ion for perchlorate analysis

PREPARATION OF TREATED DRINKING WATER:
- Dilution from a stock solution at 50 mg/mL
- Concentration adapted weekly (using body weight and water consumption) to reach the target dose-levels in mg/kg/day: 0.04-129 µg/mL
- Stirred for 30 min before storage, and again before delivery
- Frequency of preparation: at least every five weeks (stock solution), and every week (diluted solutions)
- Storage: refrigerated (diluted solutions)

Drinking water solutions containing AP were prepared on a weekly basis during the study in reverse-osmosis (RO) deionized water. The concentration of AP in the drinking water was adjusted weekly for each sex group, based on measured body weights and water consumption, to achieve the desired dosage levels.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sensitive and selective ion chromatography (IC) method for the analysis of perchlorate and nitrate, a possible interference ion, was developed by Tsui et al. (1998) to support this study. The IC method was shown to be capable of detecting both perchlorate and nitrate at 5 ppb in reagent grade water with excellent accuracy and precision. The IC method was used to verify the stability of aqueous AP solutions and to periodically confirm target concentrations of AP in the drinking water solutions prepared for the study.
The IC was performed using a dionex DX-300 High Performance Liquid Chromatograph with a Dionex CDM-3 conductivity detector. An ASRS-II anion suppresser operating in auto suppression-external mode was used. The system included a Dionex AI 350 autosampler. Anion analyses were performed using a Dionex IonPak AS-11 ion chromatography column (4.0 × 250 mm), Dionex ATC-1 anion trap column, and Dionex AG-11 guard column (4.0 × 50 mm). The mobile phase, consisting of 45 mM NaOH in 55:45 water:methanol, was set at 1 ml/min flow rate. The injection loop volume was 50 μl, and the regenerant flow rate was 10 ml/min. All IC analyses were performed at 30°C.

Duration of treatment / exposure:

- 14 days for satellite groups
- 90 days for terminal and recovery groups

Recovery: for 30 days after the 90-day treatment period.

Frequency of treatment:

Continuous (treated water ad libitum)

Remarks:

Doses / Concentrations:
0.01, 0.05, 0.2, 1 and 10 mg/kg/day
Basis:

No. of animals per sex per dose:

10M+10F (interim group) + 10M+10F (terminal group): at each dose

+10M+10F (recovery groups): at 0, 0.05, 1 and 10 mg/kg/day (there were thus in total 20M+20F treated for 90 days in these groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels for this study were selected by a panel of toxicologists representing government, academia, and industry. The goal was to select a dose range that would establish a No Observed Effect Level (NOEL) for serum hormone changes at lower doses, produce frank thyroid toxicity at higher doses, and allow identification of other possible target organ effects. Because there was little relevant literature available, the panel relied primarily on a pilot 14-day toxicity study of AP in rats by Caldwell et al. (1996).
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 30 days, in "recovery" groups treated for 90 days
- Section schedule rationale: random

Positive control:

Only for micronucleus interpretation (additional animals treated with cyclophosphamide i.p).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly, individually
- Calculated as mg/kg/day from body weight data

OPHTHALMOSCOPIC EXAMINATION: Yes. Ophthalmological examinations were performed on all animals prior to study initiation, near conclusion of treatment, and near completion of the 30-day recovery period. Eyes were dilated using 0.5% Mydriacyl® ophthalmic solution prior to bimicroscopic slit-lamp and indirect ophthalmoscopic examinations by a Board-certified veterinary ophthalmologist.
- Time schedule for examinations: day -1, day 85 and day 119.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: sacrifice (14, 90 or 120 days of study)
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 per timepoint

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: sacrifice (14, 90 or 120 days of study)
- Animals fasted: No
- How many animals: 10 per timepoint

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No (not a deviation as it was done in another study)Sacrifice and pathology

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All guideline-required organs and tissues were exmained, except for histological examination of Peyer's patches.

All animals were subjected to a complete gross necropsy examination at the time of death or scheduled euthanasia. For each rat, a complete set of tissues and organs was preserved by immersion in 10% neutral buffered formalin (NBF), including adrenals, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, exorbital lachrymal glands, eyes, larynx, femur, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mammary gland, nose, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus and vagina. Fresh organ weights were obtained for the liver, kidneys, testes, ovaries, brain, spleen, lungs, epididymides, uterus, pituitary, and heart. Following complete fixation in 10% NBF, all thyroid/parathyroid glands were carefully trimmed and weighed by a single technician.
All tissues from control and high-dose animals euthanized after 14 or 90 days were examined microscopically. Additionally, the liver, kidneys, lungs, thyroids, and gross lesions from all intermediate dose groups euthanized after 14 and 90 days and from all recovery animals were examined for histopathological changes. The tissues were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Microscopic examinations were performed by a Board-certified veterinary pathologist experienced in rodent pathology.

Other examinations:

Estrous cycling.
Vaginal smears were examined daily to assess estrous cyclicity for 3 weeks prior to scheduled euthanasia at 90 or 120 days. The smears were examined under low power magnification (10×) and classified into four stages (i.e., proestrus, estrus, metestrus, and diestrus), according to the methodology of Yuan and Carlson (1987).

Sperm analysis.
Semen samples were obtained from all male rats euthanized after 90 or 120 days for evaluation of sperm count (106/g cauda), concentration (10E6/ml), motility (%), and morphology. Sperm count and concentration were determined from samples obtained from the left cauda epididymis. Sperm motility and morphology were evaluated from samples collected from the vas deferens. A Hamilton Thorne IVOS 10 semen analyzer (Hamilton Thorne Research, Beverly, MA) was used for the sperm count, concentration, and motility assessments. Sperm morphology was assessed by microscopic examination of a minimum of 200 sperm/animal at 300–500× magnification. Morphological endpoints were based primarily on head and tail abnormalities, based on the classification systems of Linder et al. (1992) and Seed et al. (1996). The mean percentage of morphologically normal sperm was calculated for each group.

Micronucleus formation.
Bone marrow samples were collected from all animals euthanized after 90 and 120 days for possible evaluation of bone marrow micronucleus formation. Six additional (satellite) rats were administered a single intraperitoneal injection of cyclophosphamide (20 mg/kg body weight) to serve as positive controls for the micronucleus assay. Bone marrow slides from the vehicle control, positive control, and high-dose (10 mg/kg/day) groups were stained and evaluated as described previously (Schmid, 1976). The frequency of micronucleated cells was determined by random observation of 1000 polychromatic erythrocytes (PCEs) per slide. The ratio of PCEs/NCEs (normochromatic erythrocytes) was also determined to assess potential cytotoxicity due to perchlorate.

Clinical pathology and TSH, T3, and T4 analyses.
Blood samples were collected from all animals at scheduled euthanasia after 14, 90, or 120 days for evaluation of routine hematology and clinical chemistry parameters, as well as TSH, T3, and T4. The blood samples were obtained via the vena cava immediately prior to necropsy. The rats were euthanized in a randomized block design across groups to minimize any potential bias related to blood and tissue sampling times. The time of euthanasia and blood sample collection was recorded for each animal. Blood samples for hematology were analyzed using a Coulter S Plus IV hematology analyzer. Differential leukocyte counts were performed manually under oil immersion (100×) from Wright-Giemsa–stained slides. Serum samples for clinical chemistry were analyzed using a Beckman Synchron CX-5 chemistry analyzer. Appropriate controls were used to monitor the accuracy and precision of the hematology and clinical chemistry instruments.

Statistics:

Body weight, body weight gain, food consumption, water consumption, clinical pathology parameters, organ weights, estrous cycle lengths, and semen parameters were analyzed by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1967) followed by the Tukey-Kramer test (Dunnett, 1980) when appropriate. The Chi-square test (Siegal, 1956) was used to analyze the incidence of females in each group exhibiting estrous cyclicity. Serum hormone levels (TSH, T3, and T4) were statistically analyzed by ANOVA, followed by the Bonferroni multiple comparisons test (Rosner, 1990). Bone marrow micronuclei counts and PCE/NCE ratios were analyzed using ANOVA and Chi-square, respectively. All statistical comparisons utilized a minimum significance level of p < 0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs of toxicity were observed in any of the study groups and all animals survived to scheduled euthanasia following 14, 90, or 120 days, with the exception of one female in the 0.05 mg/kg/day AP group that was found dead during the recovery period. This death was considered unrelated to AP treatment, as no mortality or clinical signs of toxicity were noted at the higher AP treatment levels and the histopathologic examination for cause of death was inconclusive.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences in mean body weights among the groups. Mean body weight gain was slightly but significantly (p < 0.05) decreased in 10 mg/kg/day males during the first week. Specifically, the 10 mg/kg/day males gained an average of 57 g, whereas control males gained an average of 64 g on study week 1. Although statistically significant, this difference was not considered toxicologically meaningful. No other statistically significant differences in body weight gain were noted.

FOOD AND WATER CONSUMPTION
Occasional significant (p < 0.05) reductions in food and water consumption were observed in the AP-treatment groups during the first 3 weeks of the study (data not shown). These differences were not considered toxicologically meaningful, as they were relatively minor and they did not follow any dose-related pattern.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmological examinations did not reveal any test article–related ocular effects in the AP-treated rats. Occasional findings of slight corneal crystals and moderate conjunctival exudate were noted at study termination; however, these findings were of low incidence and randomly distributed among the groups.

HAEMATOLOGY AND CLINICAL CHEMISTRY
A few statistically significant (p < 0.05) differences in hematology and clinical chemistry parameters were observed; however, these did not follow any pattern that would indicate a relationship to AP treatment.

ORGAN WEIGHTS
Both absolute and relative thyroid weights were significantly increased (p < 0.05) in males at the 10 mg/kg/day level after 14 and 90 days and in females at the 10 mg/kg/day level after 90 days. Following the 30-day recovery period, thyroid weights of both males and females at the 10 mg/kg/day level were comparable to control rats. No other toxicologically meaningful differences in organ weights were observed.

HISTOPATHOLOGY
Routine histopathology revealed a test article–related effect in the thyroids of 10 mg/kg/day males and females after 14 or 90 days of AP treatment. The effect was characterized primarily by follicular cell hypertrophy with microfollicle formation and colloid depletion. There also appeared to be an increased number of microfollicles in the central portion of the affected thyroids; however, there was no evidence of focal hyperplasia as would be indicated by epithelial stratification or increased mitotic figures. The severity of the thyroid changes ranged from minimal to moderate; lesions of moderate severity were noted only in 10 mg/kg/day males at the 14-day interval.
In contrast to the thyroid effects noted at the 10 mg/kg/day level, no test article–related thyroid pathology was observed at AP dosage levels ≤ 1.0 mg/kg/day after 14 or 90 days of treatment. Following the 30-day recovery period, thyroids at the 10 mg/kg/day level appeared normal, with no evidence of morphological alteration.
No test article-related microscopic changes were observed in the other tissues and organs examined in this study.

SPERM ANALYSIS AND ESTROUS CYCLICITY
There were no statistically significant differences in sperm count, concentration, motility, or morphology at the end of 90 days or following the 30-day recovery period (data not shown). Similarly, no significant differences in estrous cyclicity data were noted after 90 days of treatment or following the recovery period (data not shown). The number of females cycling in each group and the mean cycle lengths remained comparable between the control and AP-treatment groups.

MICRONUCLEUS ASSAY
No test article–related changes in bone marrow micronucleus formation or PCE/NCE ratios were observed in males or females after 90 days of treatment. As expected, the positive control material, cyclophosphamide, induced a marked increase in micronucleated PCEs with no evidence of bone marrow toxicity.

Dose descriptor:

NOAEL

Remarks:

histological changes

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Follicular cell hypertrophy with microfollicle formation and colloid depletion in the rat thyroid at 10.0 mg/kg bw/d.

Critical effects observed:

not specified

TSH, T₃, and T₄analyses

After 14 days of treatment, mean TSH levels were significantly increased (p< 0.05) in males at AP levels of 0.2 mg/kg/day and higher, and in females at AP levels of 0.05 mg/kg/day and higher. The increases in TSH over control values ranged from 17% higher in the 0.05 mg/kg/day females to 62% higher in the 10 mg/kg/day males. Mean T₄levels were significantly decreased (p< 0.05) in both males and females at the 10 mg/kg/day level, with mean values 23% and 18% lower than controls, respectively. Mean T₃levels were significantly decreased (p< 0.05) in males at levels of 0.01 mg/kg/day and higher; the decreases in male T₃ranged from 21% lower than controls at the 0.01 mg/kg/day level to 39% lower than controls at the 10 mg/kg/day level. No statistically significant differences in T₃levels were observed in the female treatment groups at 14 days.

After 90 days of treatment, mean TSH levels were significantly increased (p< 0.05) in males at AP levels of 0.2 mg/kg/day and higher, and in females at the 10 mg/kg/day level only. The increases in TSH over control values ranged from 17% higher in the 0.2 mg/kg/day males to 21% higher in the 10 mg/kg/day females. Mean T₃and T₄levels were significantly decreased (p< 0.05) in both sexes at levels of 0.01 mg/kg/day and higher. The decreases in T₄ranged from 14% lower than the control group in the 0.01 mg/kg/day males to 43% lower in the 10 mg/kg/day males. The decreases in T₃ranged from 12% lower than the control group for the 0.01 mg/kg/day males to 35% lower in the 10 mg/kg/day males.

Following the 30-day recovery period, TSH levels were significantly increased (p< 0.05) in all three female recovery groups (0.05, 1.0, and 10 mg/kg/day), whereas no significant differences in TSH levels were observed in the male recovery groups. The increases in TSH levels of females ranged from 16% higher in 0.05 mg/kg/day females to 22% higher in the 10 mg/kg/day females. In contrast to the TSH results, mean T₄levels were significantly lower (p< 0.05) than controls in all three male recovery groups (0.05, 1.0, and 10 mg/kg/day), whereas no significant differences in T₄levels were observed in the female groups. The decreases in T₄levels of males ranged from 23% lower than control males in the 0.05 and 1.0 mg/kg/day groups to 39% lower than control males in the 10 mg/kg/day group. Statistically significant differences in T₃were limited to a lower mean T₃value in 10 mg/kg/day females (12% lower than control females). No statistically significant differences in T₃levels were observed in the male recovery groups.

Conclusions:

After 90-day oral (drinking water) treatment of rats with ammonium perchlorate, thyroid hormone levels were affected at low doses (T3/T4 decreases, TSH increases) but presented an unusual pattern (plateauing of amplitude + minimal TSH increase for the observed changes in T3/T4), suggesting absence of biological relevance, below 10 mg/kg/day. Non-hormonal adverse effects were only observed at 10 mg/kg/day: increased thyroid weight and thyroid follicle hypertrophy.

Executive summary:

Ammonium perchlorate was provided to groups of 10 male and 10 female rats via drinking water, at dose-levels of 0 (control), 0.01, 0.05, 0.2, 1 and 10 mg/kg/day for 14 and 90 days. Additional 10 rats per sex were treated in the same way at 0, 0.05, 1 and 10 mg/kg/day and kept for a 30-day treatment-free period. Data for the 14-day treatment are ignored here.

At 10 mg/kg/day, the only toxicologically relevant effects were related to thyroids. Thyroids were of higher absolute and relative weights than in controls, with follicle hypertrophy. All these changes were completely reversible in 30 days. Marked thyroid hormone changes were noted in both sexes. Serum levels of T3 and T4 were significantly lower than in controls (-28% to -43%), respectively with almost complete (-9% to -12%) and incomplete (-11% to -31%) recoveries. Serum levels of TSH were significantly higher than in controls (+18% to +21%), with no clear recovery (+9% to +22%). At 0.05 to 1 mg/kg/day, poorly dose-related (plateauing) effects on thyroid hormones occured: T3 and T4 reductions (-16% to -32%) and minimal TSH increases (at most +18%), which seems not coherent with the usual high compensation by TSH for moderate changes in T3/T4. The biological relevance seemed therefore low. Recovery was evidenced for T3, T4 and TSH.

At 0.01 mg/kg/day, T3 and T4 were still significantly lower (-12% to -20%) while TSH was unaffected. Recovery was not assessed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study, OECD 408 compliant.

Additional information

In a study (Siglin, 2000), Ammonium perchlorate was provided to groups of 10 male and 10 female rats via drinking water, at dose-levels of 0 (control), 0.01, 0.05, 0.2, 1 and 10 mg/kg/day for 14 and 90 days. Additional 10 rats per sex were treated in the same way at 0, 0.05, 1 and 10 mg/kg/day and kept for a 30-day treatment-free period.

After 90-day oral (drinking water) treatment of rats with ammonium perchlorate at dose-levels of 0, 0.01, 0.05, 0.2, 1 and 10 mg/kg/day, thyroid hormone levels were affected at low doses (T3/T4 decreases, TSH increases) but presented an unusual pattern (plateauing of amplitude + minimal TSH increase for the observed changes in T3/T4), suggesting absence of biological relevance, below 10 mg/kg/day. Non-hormonal adverse effects were only observed at 10 mg/kg/day: increased thyroid weight and thyroid follicle hypertrophy.

These changes were reversible after a 30 days recovery period.

As compared to humans, the rat is known to be more sensitive to perturbations of the pituitary-thyroid axis. This greater sensitivity is related to shorter plasma half-lives of T₃ and T₄ in the rat and to differences in T₃transport proteins between rats and humans (Capen, 1997). Both rodents and humans have nonspecific low-affinity protein carriers of thyroid hormones (e.g., albumin). However, humans also possess a high-affinity binding protein, thyroxine-binding globulin, that is virtually undetectable in adult rats (Vranckxet al., 1994). Because this protein is missing in adult rats, more T₄ is susceptible to removal from the blood and hence to metabolism and excretion. Consequently, the serum half-life of T₄ in rats (< 1 day) is substantially shorter than in humans (approximately 5 to 9 days). It has been estimated that this difference results in a 10-fold greater requirement for exogenous T₄ in the rat with a non-functioning thyroid compared to the adult human (Döhleret al., 1979).

Perchloric acid, once absorbed in the general circulation is expected to be transformed into perchlorate moiety because of the blood buffering effect. Therefore the results of this study on ammonium perchlorate (CAS no. 7790-98-9) are used in a read across approach to evaluate perchloric acid repeated exposure toxicity.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available, used for read-accross

Justification for classification or non-classification

In the available study on Ammonium perchlorate specific effects on the thyroid have been evidenced with a NOAEL of 1mg/kg/day on rats but with absence of any other effect on traditional indicators like body weight, clinical signs, clinical chemistry and histopathology. However, rats are know to be more sensitive to thyroid inhibition of iodine intake by an estimated ten fold factor. Moreover these effects are fully reversible and perchlorate is not bioaccumulable.

Based on these results Perchloric acid is classified as harmful by repeated oral administration according to DSD 67/548/EC and Single Target Organ Toxicant by repeated administration category 2 for the thyroid according to CLP 1272/2008/EC criteria.