Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 1997 - April 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Sufficiently compliant to GLP. Wrong OECD guideline quoted. Occasional inconsistence between data, calculations, comments and conclusions (e.g. drinking water perchlorate concentrations wrongly stated to be within +-10%; errors in gestation index calculations). The top-dose was not based on dose-limiting toxicity but on antithyroid effects, limiting the sensitivity of the study for classification for effects on Fertility. Some investigations are lacking or are not performed according to the most sensitive method. Thyroid adenomas not reported in the report conclusion.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
not specified
Remarks:
not examined; see deviations to OECD method
Qualifier:
according to
Guideline:
other: EPA Pesticides Assessment Guidelines. Subdivision F
Deviations:
not specified
Remarks:
not examined; see deviations to OECD method
Qualifier:
according to
Guideline:
other: JMAFF. 59 NohSan No. 4200
Deviations:
not specified
Remarks:
not examined; see deviations to OECD method
Qualifier:
according to
Guideline:
other: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not applicable
Remarks:
This guideline is not applicable to this test (mistake in report)
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
NB: guideline not cited in report.
Deviations:
yes
Remarks:
Testes stained with hematoxylin/eosin. Uterus+coagulating glands not weighed. No histology of primordial follicles. Paternal/maternal NOAELs instd. of parental/offspring NOAELs (set by RSS author). T°C: 18-26 instd. of 19-25.
GLP compliance:
yes
Remarks:
No laboratory compliance certificate. No test item analytical certificate. Perchlorate concentrations were most often outside the range validated for stability at the highest dose.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: MCP
- Physical state: solid powder
- Nominal purity: not indicated (NB: 99.8% based on other studies on the same batch)
- 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

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: (P) more than 5 wks; (F1) 3 wks (once weaned)
- Weight at study initiation: (P) Males: 112-144 g; Females: 121-145 g; (F1) means: 43-46 g (once weaned)
- Fasting period before study: no
- Housing: cages not detailed, individual except during mating and lactation (females)
- Diet (e.g. ad libitum): PMI Certified Rodent Diet, ad libitum
- Water (e.g. ad libitum): reverse osmosis water, ad libitum
- Acclimation period: 6 days

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

IN-LIFE DATES: From: 1998-01-06 To: 1998-09-16

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on 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
- Less than 0.5 mg/L (LOD) of nitrate, a possible interference ion for perchlorate

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: 0.96-526 µg/mL
- Frequency of preparation: at least once a week (diluted solutions)
- Storage: refrigerated (stock solutions) / not indicated (diluted solutions)
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear, referred to as day 0 of pregnancy
- No replacement of males or further mating.
- Verification of same strain and source of both sexes: yes
- After successful mating each pregnant female was caged (how): in a nesting box
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 all doses: concentrations within +/- 10% of nominals on 8/9 preparation occasions, except for preparations dated 5 may (up to +49%).
- at 0.05 and 200 µg/mL: stable over 109 days at 21-22°C under light (12h/day) - demonstrated in another study (7.5.1)

Comments:
- For almost all high-dose preparation occasions, the target concentrations were above the maximal concentration demonstrated to be stable (200 µg/mL).
- This validation otherwise adequately covers the study's test item use conditions.
Duration of treatment / exposure:
P1: 113 to 137 days
F1 adults: 125 to 142 days
(incl. at least 70 days before mating, up to 2-week mating, 3-week gestation, 3-week lactation)
Frequency of treatment:
Continuous (treated water ad libitum)
Details on study schedule:
- F1 parental animals not mated until 70 days of exposure after selected from the F1 litters.
- Selection of parents from F1 generation when pups were weaned/21 days of age.
- Age at mating of the mated animals in the study: at least 15 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.3 mg/kg/day
Basis:
other: nominal dose; time-weighed average achieved dose within +/- 10% of nominal, except during gestation for P1 females (+33%) and F1 females (-33% ), and during lactation of F1 females (+33%)
Remarks:
Doses / Concentrations:
3 mg/kg/day
Basis:
other: nominal dose; time-weighed average achieved dose within +/- 10% of nominal, except during gestation for P1 females (+33%) and during lactation of F1 females (+20%)
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
other: nominal dose; time-weighed average achieved dose within +/- 10% of nominal, except during gestation for P1 females (+34%) and during exposure of F1 males (-12%) and during lactation of F1 females (+16%)
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 14-day study with NOAELs of 0.44(M) / 0.12(F) mg/kg/day, on a neurobehavioral study, and on a 90-day study (see 7.5.1)
- Rationale for animal assignment: random
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly throughout study

FOOD CONSUMPTION: Yes
- Time schedule: weekly throughout study

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: at least 3 times a week, individually
- Calculated as mg/kg/day from body weight data

THYROID HORMONE LEVELS:
TSH, T3 and T4 in serum at sacrifice after weaning of pups
Oestrous cyclicity (parental animals):
Assessed daily (vaginal cytology) during 3 weeks before mating, and during mating until evidence of successful mating
Sperm parameters (parental animals):
Parameters examined in P/F1 male parental generations:
testis weight, epididymis weight, prostate weight, seminal vesicle weight, spermatid count in testes, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS: not indicated, but there were at most 30 rats/sex/dose/offspring generation

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
- evaluation of vital status at birth (to identify stillborn pups)
- gross exam. for external and internal abnormalities; possible cause of death was determined for pups born or found dead

THYROID HORMONE LEVELS:
TSH, T3 and T4 in serum at sacrifice of pups after weaning
Postmortem examinations (parental animals):
SACRIFICE
- Male and female animals: all surviving animals after the last litter of each generation was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs/tissues were prepared for microscopic examination and weighed as required by OECD 416, except for those listed in deviations.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age (weaning).
- 3 pups/sex/litter/generation were subjected to postmortem macroscopic and microscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS
Organs/tissues were prepared for microscopic examination and weighed as required by OECD 416, except for those listed in deviations. Thyroids/parathyroids were also weighed.
Statistics:
See page 54 of report.
Reproductive indices:
Mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

Fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

Gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females
Offspring viability indices:
Live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

Viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups

Lactation index on day 21 post-partum:
Number of surviving pups on day 21 post-partum
________________________________________ x 100
Number of surviving pups on day 4 post-partum

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL/ADULT ANIMALS)
P1: One male at 30 mg/kg/day and one female at 3 mg/kg/day sacrificed as the result of an injury.
F1: Three males and one female at 30 mg/kg/day were found dead, the cause was not established but considered unrelated to treatment.

TREATED DRINKING WATER INTAKE (PARENTAL/ADULT ANIMALS)
Reduced in P1 males at 30 mg/kg/day from day 36 until sacrifice. Treatment-related, but not toxicologically relevant (not confirmed with dose-relationship in P1 females, F1 males and F1 females).

THYROID HORMONE LEVELS (PARENTAL/ADULT ANIMALS)
P1: Serum T4 levels significantly decreased and TSH levels significantly increased at 30 mg/kg/day in males. Females unaffected.
T3 significantly increased in males at 0.3, 3.0 but not 30 mg/kg/day: not considered toxicologically relevant (opposite effect when compared to anti-thyroid effects of perchlorate).
F1: Significant increase in TSH (M+F) and decrease in T4 (M) at 30 mg/kg/day: toxicologically relevant. Additionally in males, increased T4 at 0.3 and 3 mg/kg/day: not considered toxicologically relevant (opposite effect when compared to anti-thyroid effects of perchlorate).

ORGAN WEIGHTS (PARENTAL/ADULT ANIMALS)
P1: Significantly higher absolute and/or relative thyroid weights at 3 (M) and 30 (M+F) mg/kg/day.
F1: Significantly higher absolute and relative thyroid weights at 0.3 mg/kg/day in F: not biologically relevant when compared with P1 data; and also at 3 (M+F) and 30 (M+F) mg/kg/day (relevant). Increased spleen weight in males, not toxicologically relevant (not confirmed with dose-relationship and significance in any other generation or sex).

HISTOPATHOLOGY (PARENTAL/ADULT ANIMALS)
P1,F1: dose-related increase in incidence and severity of follicular epithelium hypertrophy and hyperplasia in thyroid, statistically significant from 3 mg/kg/day.
F1: 2 of the 30 male rats developed thyroid follicular cell adenoma by 19 weeks of age, at 30 mg/kg/day. Considered treatment-related by RSS author.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
0.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Relevant antithyroid effects in P1 and F1 adults from 3 mg/kg/day.
Remarks on result:
other: Generation: P1 and F1 adults (migrated information)
Dose descriptor:
NOAEL
Remarks:
Offspring toxicity
Effect level:
0.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Revised by summary author (original report: 3 mg/kg/day). Based on antithyroid effects at higher doses.
Remarks on result:
other: Generation: F1 and F2 pups (migrated information)
Dose descriptor:
NOAEL
Remarks:
Reproduction toxicity
Effect level:
>= 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of effects towards reproduction at up to the maximal tested dose.
Remarks on result:
other: Generation: all generations (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related

Details on results (F1)

THYROID HORMONE LEVELS (OFFSPRING)
F1: Significant reduction in T3 in females at 30 mg/kg/day: toxicologically relevant. Reduction in TSH and increase in T4 at 0.3 and/or 3.0 but not 30 mg/kg/day in males, not considered toxicologically relevant (opposite effect when compared to anti-thyroid effects of perchlorate).
F2: Reduction in T3 levels, although not significant, at 30 mg/kg/day in females.

ORGAN WEIGHTS (OFFSPRING)
F1: Significantly higher absolute thyroid weights at 30 mg/kg/day (M+F); relative weights not reported.
F1: In females, higher absolute thyroid weights at 30 (significant) and 3 mg/kg/day (not significant); relative weights not reported.

HISTOPATHOLOGY (OFFSPRING):
F1,F2: dose-related increase in incidence and severity of follicular epithelium hypertrophy and hyperplasia in thyroid, statistically significant from 3 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Effects in adult rats (P1 and F1 adults) exposed to ammonium perchlorate

Sex Male Female
Dose-level (mg/kg/day) 0 0.3 3.0 30 0 0.3 3.0 30
P1 adults at sacrifice (113 -137 days of treatment)
Serum T4 level (µg/dL) 4.64 4.73 4.74 3.58** not affected
Serum TSH level (ng/mL) 1.53 1.35 1.49 3.87** not affected
Thyroid weight (absolute, mg) 39 41 43* 51** 24 25 25 30**
Thyroid weight (% relative to brain) 1.67 1.72 1.81 2.17** 1.10 1.15 1.13 1.37**
Follicle hypertrophy@ 16/30 12/30 22/30 24/30 2/27 3/21 13/26 17/24
Follicle hyperplasia@ 2/30 1/30 5/30 8/30 0/27 0/21 0/26 6/24
F1 adults at sacrifice (125 -142 days of treatment)
Serum T4 level (µg/dL) 3.78 4.21* 4.20* 2.78** not affected
Serum TSH level (ng/mL) 2.51 2.16 2.30 5.18** 1.62 1.22 1.65 2.12*
Thyroid weight (absolute, mg) 36 41 44** 63** 22 25* 28** 33**
Thyroid weight (% relative to brain) 1.52 1.68 1.77** 2.64** 1.01 1.13* 1.28** 1.51**
Follicle hypertrophy@ 8/29 8/30 16/30 27/30 2/20 4/27 5/27 12/23
Follicle hyperplasia@ 5/29 5/30 11/30 13/30 1/20 1/27 3/27 0/23
Follicle adenoma (number of rats)@ 0/29 0/30 0/30 2/30 0/20   0/27  0/27  0/23
Follicle adenoma (number of adenomas)@  0 0 0 3 0 0 0 0

statistical difference from controls: *: p<0.05; **: p<0.01; @: peer-review data, no statistical analysis performed

Effects in rat pups (F1 and F2 weaned pups) exposed to ammonium perchlorate

Sex Male Female
Dose-level (mg/kg/day) 0 0.3 3.0 30 0 0.3 3.0 30
F1 pups at weaning
Serum T3 level (ng/dL) not affected 106 110 109 97.6*
Thyroid weight (absolute, mg) 8 8 8 10** 8 9 8 10**
Follicle hypertrophy@ 2/28 4/22 14/25 23/23 3/28 7/22 15/24 21/21
Follicle hyperplasia@ 0/28 0/22 5/25 6/23 0/28 1/22 1/24 7/21
F2 pups at weaning
Serum T3 level (ng/dL) not affected 108 107 108 98.8
Thyroid weight (absolute, mg) not affected 8 8 10 9*
Follicle hypertrophy@ 0/20 0/27 4/28 7/25 0/20 1/27 8/28 13/25
Follicle hyperplasia@ 0/20 0/27 0/28 0/25 0/20 0/27 2/28 1/25
statistical difference from controls: *: p<0.05; **: p<0.01; @: peer-review data, no statistical analysis performed

Applicant's summary and conclusion

Conclusions:
Parental NOAEL was 0.3 mg/kg/day due to antithyroid effects. However, the parental MTD was above 30 mg/kg/day in the absence of relevant general toxicity (excluding antithyroid effects).

Offspring NOAEL was 0.3 mg/kg/day, as opposed to original report conclusion (3 mg/kg/day), based on anti-thyroid effects at higher doses.

Reproduction NOAEL set at 30 mg/kg/day in the absence of effects on reproductive function at the doses investigated.

The top-dose was not based on dose-limiting toxicity but on antithyroid effects, limiting the sensitivity of the study for classification for effects on Fertility.
Executive summary:

A two-generation study was conducted in rats exposed to ammonium perchlorate at 0, 0.3, 3.0 and 30 mg/kg/day.

  • At 30 mg/kg/day, there was a clear antithyroid effect in P1 and F1 adults and F1 and F2 pups: lower serum T3 (female pups) or T4 levels (male adults), higher serum TSH level (adult P1 males, adult F1 males and females), higher absolute and (when determined) relative thyroid weights (in all conditions), minimal to marked thyroid follicle hypertrophy and hyperplasia (in all conditions), thyroid follicle adenomas in two males. There was no general parental toxicity: the maximal tolerated dose was not reached, limiting sensitivity of the study according to summary author. No effects on reproduction and in particular fertility and pup growth were noted.
  • At 3 mg/kg/day, no relevant hormonal effects were noted; thyroid weight was affected in adults only; however, incidence and severity of thyroid follicle lesions was increased in all conditions (except possibly in F1 adult females were it was not obvious).
  • At 0.3 mg/kg/day, no relevant hormonal effects were noted; thyroid weight was minimally increased in F1 adult females but this was considered not biologically relevant; no relevant effect on thyroid lesions.