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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1997 - June 1998
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. The few deviations have no impact on the reliability and completeness of the conclusions. Histopathological examinations did not distinguish between hypertrophy and hyperplasia, but this was done later by peer-review.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Temperature (°C): 18-26 instead of 19-25. Highest dose chosen with the aim to induce antithyroid effects, but not based on dose-limiting toxicity. No histological examination of Peyer’s patches.
GLP compliance:
yes
Remarks:
No laboratory compliance certificate. No analytical certificate for test item. Perchlorate concentrations were occasionally outside the range validated for stability.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: MCP
- Physical state: solid powder
- Nominal purity: 99.8% (not verified)
- Lot/batch No.: 03907LF
- Expiration date of the lot/batch: not indicated
- Stability under test conditions: not indicated but stability verified in drinking water
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6-7 weeks (indicated only in published version of report)
- 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): PMI Certified Rodent Chow, ad libitum
- Water (e.g. ad libitum): reverse osmosis water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1997-9-9 To: 1998-1-9

Administration / exposure

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
- Lot/batch no. (if required): not applicable
- Purity: not applicable
- 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)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Methods:
- validated ion chromatography method
- LOD for perchlorate and nitrate (interference ion): 0.005 µg/mL

Results:
- at 0.05 and 200 µg/mL: concentrations within +/- 10% of nominals
- at 0.05 and 200 µg/mL: stable over 109 days at 21-22°C under light (12h/day)

Comment:
- For the first of 5 concentration measurement dates, group 3 was exposed to lower drinking water perchlorate (nominal and achieved) levels than group 2: it was likely a 10-fold too low nominal concentration setting by error (0.04 µg/mL instead of > 0.4 µg/mL at other preparation dates). Furthermore, this low level was below the minimal concentration demonstrated to be stable (0.05 µg/mL).
- This validation otherwise adequately covers the study's test item use conditions.
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)
Doses / concentrations
Remarks:
Doses / Concentrations:
0.01, 0.05, 0.2, 1 and 10 mg/kg/day
Basis:
other: nominal dose; achieved dose within +/- 10% of nominal
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: panel of toxicology experts, focused on antithyroid effects, to identify doses with frank effects and a possible NOEL
- 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.)

Examinations

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
- 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:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All guideline-required organs and tissues were examined, except for histological examination of Peyer's patches.
Other examinations:
- Estrous cycles, all females daily during 3 weeks before sacrifices at 90 and 120 days*
- Sperm analyses, all males at 90 and 120-day sacrifices*
- Bone marrow micronucleus evaluation, rats at 0 and 10 mg/kg/day and positive controls, at 90 and 120-day sacrifices (NB: does not follow requirements of an in vivo micronucleus study)*
*: low-detail sufficient as specific, more sensitive studies cover the endpoints fertility and mutagenicity

- Serum hormone levels: TSH, T3, T4 in all rats of all groups at 14, 90 and 120 days.
Statistics:
Continuous variables: one-way ANOVA + Tukey-Kramer intergroup comparisons
Chi-square for % females cycled.
All tests at significance level of 5%.

Results and discussion

Results of examinations

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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY:
One female at 0.05 mg/kg/day died during the recovery period. The cause could not be determined, but was not considered to be related to treatment.

BODY WEIGHT GAIN
Slightly lower in males at 10 mg/kg/day over the first week only. Not considered toxicologically relevant.

FOOD CONSUMPTION
Slightly reduced in all treated groups over first 3 weeks only. Not considered toxicologically relevant.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Consumption reduced in all treated groups at some occasions during first 3 weeks. Not considered toxicologically relevant.

HAEMATOLOGY
Decreased monocytes at 0.05 and 0.2 mg/kg/day in females at 90 days. Not considered toxicologically relevant.

CLINICAL CHEMISTRY
Some slight changes in 0.01 mg/kg/day males at 90 days. Not considered toxicologically relevant.

ORGAN WEIGHTS
Higher absolute and relative thyroid (weighed with parathyroid) weights at 10 mg/kg/day at 14 days (males only) and 90 days (both sexes). Full recovery in the recovery group.
Some changes in absolute but not relative testes and pituitary weights, without lesions. Not considered toxicologically relevant.

GROSS PATHOLOGY
Reddened thyroids in 3/10 males at 10 mg/kg/day at 90 days.

HISTOPATHOLOGY: NON-NEOPLASTIC
Increased incidence of follicular cell hypertrophy and hyperplasia (based on peer-review) in thyroids of both sexes at 14 and 90 days, at 10 mg/kg/day. Generally minimal.

OTHER FINDINGS
- Estrous cycles: no effect.
- Sperm analyses: no effect.
- Bone marrow micronucleus: neither myelotoxic nor genotoxic effects (NB: cyclophosphamide: genotoxicity, without myelotoxicity)
- Thyroid hormone levels: KEY EFFECTS: dose-related reduction in T3 and T4 and increase in TSH, see table below. They occurred at low doses 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. All effects were reversible.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Hormonal effects (T3/T4 reductions, TSH increase) occur at low doses but present an important plateauing of amplitude, suggesting absence of biological relevance, below 10 mg/kg/day. All effects were reversible.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mean thyroid hormone level values in adult rats

 

 At 90 days of treatment   

At 120 days (30-day recovery)

Dose-level (mg/kg/day)

0

0.01

0.05 

 0.2

 1

 10

 0

 0.05

 1

 10

T3 (ng/dL) - males

180

158*

125***

123***

122***

117***

203

214

212

185

T3 (ng/dL) - females

170

143**

143**

137***

123***

122***

224

216

215

197*

T4 (µg/dL) - males

5.1

4.4**

3.7***

3.5***

3.5*** 

2.9***

 4.9

4.0***

 4.0***

3.4***

T4 (µg/dL) - females

4.4

3.5***

3.4***

3.4***

3.2***

3.0***

3.5

3.4

3.3

3.1

TSH (ng/mL) - males

 16.2

16.9

17.8

19.0**

19.1**

19.1**

20.9

21.3

22.2

22.8

TSH (ng/mL) - females

 16.5

16.8

16.8

17.4

17.7

20.0***

 13.1

15.5**

15.8**

16.0***

*: p<0.05; **: p<0.01; ***: p<0.001; N= 9-10 rats/sex/dose/timepoint

 

Incidence of histopathological thyroid follicle lesions in adult rats (peer-reviewed data)

 

 At 90 days of treatment

At 120 days (30-day recovery)

Dose-level (mg/kg/day)

0

0.01

0.05 

 0.2

 1

 10

 0

 0.05

 1

 10

Hypertrophy - males

1/10

2/10

0/10

2/10

3/10

8/10

2/10

4/10

2/10

0/10

Hypertrophy - females

0/10

0/10

3/10

2/10

1/10

5/10

0/10

0/10

1/10

0/10

Hyperplasia - males

0/10

0/10

0/10

0/10

0/10

4/10

1/10

3/10

1/10

0/10

Hyperplasia - females

0/10

0/10

0/10

0/10

0/10

3/10

0/10

0/10

0/10

0/10

no statistical analysis performed

Applicant's summary and conclusion

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: thyroid redness, increased thyroid weight and thyroid follicle hypertrophy and minimal hyperplasia.
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 reddened at 90 days (3/10 males) and of higher absolute and relative weights than in controls, with follicle hypertrophy and hyperplasia (generally minimal). 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 occurred: 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.