Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is given in section 13 of IUCLID.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

U.S. EPA GLP regulations 40 CFR Part 160

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium perchlorate

EC Number:

232-235-1

EC Name:

Ammonium perchlorate

Cas Number:

7790-98-9

Molecular formula:

ClHO4.H3N

IUPAC Name:

Ammonium perchlorate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR VAF/Plus

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: appr. 7 weeks old.
- Housing: individually, in suspended stainless steel cages.
- Diet: Purina Certifies Roden Chow (PMI Rodent Meal #5002, Purina Mills, Inc) ad libitum.
- Water: ad libitum. The drinking water was provided in individual water bottles to allow measurement and test substance consumption.
- Acclimation period: 2 weeks approximately.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C.
- Relative humidity: 50 ± 15 %.
- Air changes: 12-15 air changes/hr.
- Photoperiod: 12 hrs dark / 12 hrs light.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: drinking water solutions containing the test substance were prepared on a weekly basis during the study in reverse osmosis (RO) deionized water. The concentration of the substance in 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 was used to verify the stability of aqueous test solutions and to periodically confirm target concentrations of the substance in the drinking water solutions prepared. Furthermore, IC method was used for the analysis of nitrate, a possible interference ion: IC method was capable of detecting both ions at 5 ppb in reagent grade water with excellent accuracy and precision.

DETAILS ON ANALYTICAL METHOD
- IC: Dionex DX-300 HPLC with a Dionex CDM-3 conductivity detector. An ASRS-II anion suppresser operating in auto suppresion-external mode was used.
- Column: Dionex IonPak AS-11 ion chromatograohy column (4.0x250 mm), Dionex ATC-1 anion trap column and Dionex AG-11 guard column (4.0 x 50 mm).
- Mobile phase: 45 mM NaOH in 55:45 water:methanol.
- Flow rate: 1 ml/min.
- Regenerant flow rate: 10 ml/min.
- Injection: 50 μl.
- Temperature: 30 °C.

Duration of treatment / exposure:

14 days, 90 days.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.01 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

0.05 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

0.2 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the goal was to select a dose range that would establish No Observed Effect Level (NOEL) for serum hormone levels at lower doses, produce frank thyroid toxicity at higher doses, and allow identification of other possible target organ effects. As there was little relevant literature available, the panel relied priarily on a pilot 14-day toxicity study of the substance in rats by Caldwell et al. (1996)
- Post-exposure recovery period: an additional 10 animals/sex in groups 0, 0.05, 1.0 and 10.0 mg/kg/day were observed after a nontreatment recovery period of 30 days.

Calwell, D.J., Kinkead, E.R., Wolfe, R.E., King, J.H., Narayanan, L., Confer, P.D., and Mattie, D.R. (1996). The Toxicologist 30 (1), 67.

Examinations

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: daily.

CLINICAL SIGNS OF TOXICITY: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Frequency of examinations: weekly.

FOOD CONSUMPTION: Yes.
- Frequency: weekly.

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly.

OPTHALMOSCOPIC EXAMINATION: Yes. Eyes were dilated using 0.5 % Mydriacyl opthalmic solution prior to bimicroscopic slit-lamp and indirect opthalmoscopic examinations by a Board -certifies veterinary opthalmologist.
- Time schedule for examinations: prior to study initiation, near conclusion of treatment, and near completion of the 30-day recovery period.
- Dose groups that were examined: all aninals.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes.
- Collection of blood: at scheduled euthanasia after 14, 90, or 120 days. The blood samples were obtained via the vena cava immdiately prior to necropsy. The time of blood sample collection was recorded for each animal.
- Animals fasted: not specified.
- Dose groups that were examined: all animals.
- Parameters examined: blood samples for hematology were analyzed using a Coulter S Plus IV hematology analyzer. Differential leukocyte counts were performed manually under oil immersion (100x) 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.
- Determination of TSH, T3 and T4 levels: for each animal, serum for TSH, T3 and T4 determination was divided into three vials (500 μl/vial), which were immediately frozen and stored at -70 °C until analysis. The hormone assays were conducted using radio-immunoassay (RIA) kits, following the manufacturer's instructions. For each hormone evaluated, RIA kits with the same lot number and expiration date were used. Tracer (125I-) radioactivity was measured using a gamma counter, RIA kits (T3 standars lot numbers C30-3 to C30-8; 125I-T3 lot #TT42-0241) were purchased from Diagnostic Product Corp. and rabbit T4 antibody tubes (lot #TT41-1200) were used. TSH RIA kits (assay code #RPA554) were purchased from Amsterdam Corp. and lyophilized rabbit anti-rat TSH serum and Amerlex-M second antibody (donkey anti-rabbit serum coated onto magentized polymer containing sodium azide) were both used. All samples and standards were analyzed in triplicate.

ESTROUS CYCLING: Yes
- Time schedule: vaginal smears were examined daily to assess estrous cyclicity for 3 weeks prior ro scheduled euthanasia at 90 or 120 days.
- Examinations: the smears were examined under low power magnification (10x) and classified into four stages (proestrus, estrus, netestrus, diestrus).

SPERM ANALYSIS: Yes
- Time schedule: semen samples were obtained from all male rats euthanised after 90 or 120 days.
- Examinations: the semen samples were evaluated for sperm count (10^6/g cauda), concentration (10^6/ml),motility (%) and morphology. Sprem count and concentration were determined from samples obtained from the left cauda epididymis. Sperm mobility and morphology were evaluated from samples collected from the vas deferens. A Hamilton Thorne IVOS 10 semen analyzer 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 x 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.

OTHER:
- 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 (satellie) rats were administered a single intraperitoneal injecion of cyclophosphamide (20 mg/kg bw) to serve as positive controls for the micronucleus assay. Bone marrow slides from the vehicle control, positive control and high-dose groups were stained and evaluated. 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 the substance.

Linder, R.E., Strader, L.F., Slott, V.L. and Suarez, J.D.(1992). Reprod. Toxicol. 66, 491-505.
Seed, J., Chapin, R.E., Clegg, E.D., Dostal, L.A., Foote, R.H., Hurtt, M.E., Klinefelter, G.R., Makris, S.L., Perrault, S.D., Schrader, S., Seyler, D., Sprando, R., Treinen, K.A., Veermachaneni, D.N.R., and Wise, L.D. (1996). Reprod. Toxicol. 10 (3), 237-244.

Sacrifice and pathology:

SACRIFICE AND NECROPSY
- Ten animals/sex/group were euthanised and necropsied after 14 and 90 days of treatment.
- An additional 10 animals/sex in groups 1 (vehicle control), 3 (0.05 mg/kg bw/day), 5 (1.0 mg/kg bw/day) and 6 (10.0 mg/kg bw/day) were designated for euthanasia after a nontreatment recovery period of 30 days.
- All surviving rats were euthanised by CO2 inhalation followed by exsanguination. The time of euthanasia was recorded for each animal. The rats were euthanised in a randomized block design across groups to minimize any potenrial bias related to blood and tissue samppling times.

GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes. 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 were preserved by immersion in 10 % neutral buffered formalin (NBF), incuding adrenals, aorta, brain, cerum, colon, duodenum, epidymides, esophagus, exorbital lachrymal glands, eyes, larynx, femur, gross lessions, heart, ileum, jejenum, kideneys, 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.
-Organ weighing: 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.
- Histopathology: all tissues from control and high-dose animals euthanized after 14 or 90 days were examined microscopically. Additonally, the liver, kidneys, lungs, thyroids, and gross lessions from all intermediate dose groups euthanised 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.

Statistics:

Body weight, body weight gain, food consumption, water consumption, clinical pathology parameters, organ weights, estrous cycle lengths and semen parameters were analyzed by ANOVA followed by the Tukey-Kramer test when appropriate; The Chi-square test was used to analyze the incidence of females in each group exhibiting estrous cyclicity; Serum hormone levels were statistically analyzed by ANOVA, followed by the Bonferroni multiple comparisons test; 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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 0.05 mg/kg/day group was found dead during the recovery period. This death is considered unrelated to the substance treatment, as no mortality or clinical signs of toxicity were noted at the higher treatment group and the histopathologic examination for cause of death was inconclusive.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 signifiant differences in body weight gain were noted.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Occasional significant reductions (p<0.05) in food consumption were observed in the substance-treated groups during the first 3 weeks of the study. These differences were not considered toxicologically meaningful, as they were relatively minor and did not follow any dose-pattern.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Occasional significant reductions (p<0.05) in water consumption were observed in the substance-treated groups during the first 3 weeks of the study. These differences were not considered toxicologically meaningful, as they were relatively minor and did not follow any dose-pattern.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related occular effects in the treated groups.
Occasional findings of slight corneal crystals and moderate conjuctival exudate were noted at study termination; however these findings were of low incidence and randomly distributed among the groups.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant (p<0.05) differences in hematology parameters were observed; however these did not follow any pattern that would indicate a relationship to the treatment. The monocytes level in females of 0.05 and 0.2 mg/kg/day, were significantly different from the control group (p<0.05). Hematology parameters values in male groups were not significantly different from the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant (p<0.05) differences in clinical chemistry parameters were observed; however these did not follow any pattern that would indicate a relationship to the treatment. The sorbitol dehydr. and potassium levels in males of 0.01 mg/kg/day, were significantly different from the control group (p<0.05). No values in female groups were significantly different from the control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The severity of the thyroid changes ranged from minimal to moderate; lessions of moderate severity were noted only in 10 mg/kg/day males at the 14-day interval. When the thyroid slides were reexamined in a blind fashion, a few of the high-dose thyroids could not be distinguished from controls, illustrating the minimal natura of the change in most high-dose animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test article-related effect in the thyroids of 10 mg/kg/day males and females after 14 or 90 days of treatment. No test-article thyroid pathology was observed at 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.
The effect on thyroid was characterised by follicular cell hypertrophy with microfollicle formation and colloid depletion. An increased number of microfollicles in the central portion of the affected thyroids was also observed. However there was no evidence of focal hyperplasia as would be indicated by epithelial stratification or increased mitotic figures.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Hormone levels (TSH, T3, T4) were significantly different than the ones in control groups.

Details on results:

HORMONE LEVELS (TSH, T3 and T4)
14-DAYS TREATMENT
- Mean TSH levels were significantly increased (p<0.05) in males at treatment levels of 0.2 mg/kg/day and higher and in females at treatment levels of 0.05 mg/kg/day and higher. The increases in TSH over control ranged from 17 % higher in the 0.05 mg/kg/day females to 62 % higher in the 10 mg/kg/day males.
- Mean T4 levels were significantly decreased in both males (mean values 23 % decrease) and females (mean values 18 % decrease) at 10 mg/kg/day.
- Mean T3 levels were significantly decreases in males at levels of 0.01 mg/kg/day and higher. No statistically significant differences in T3 levels were observed in females after treatment for 14 days.
90 DAYS TREATMENT
- Mean TSH levels were significanlty increased in males at treatment kevels of 0.2 mg/kg/day and higher, and in females at the 10 mg/kg/day 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 T3 and T4 levels were significantly decreased in both sexes at levels of 0.01 mg/kg/day and higher. The decreases in T4 ranged 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 T3 ranged 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.
30-DAY RECOVERY PERIOD
- TSH levels were significanlty increase in all three female recovery groups (0.05, 1.0 and 10 mg/kg/day). 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. No significant differences in TSH levels were observed in the male recovery groups.
- Mean T4 levels were significantly lower than controls in all three male recovery groups (0.05, 1.0 and 10 mg/kg/day). The decreases in T4 levels of males ranged from 23 % lower than control males in 0.05 and 1.0 mg/kg/day to 39 % lower thsn control males in the 10 mg/kg/day. No significant differences in T4 levels were observed in the female groups.
- Statistically significant difference in T3 were limited to a lower mean T3 value in 10 mg/kg/day females (12 % lower than control females). No statistically significant differences in T3 levels were observed in the male recovery groups.

Despite the fact that mean TSH levels were significantly increased in males at levels of 0.2 mg/kg/day and above and in females at levels of 0.05 mg/kg/day and above, there were no indications of a thyroid growth response at perchorate levels ≤ 1.0 mg/kg/day. The increases in TSH at levels ≤ 1.0 mg/kg/day were not sufficient to induce an observable growth response in the rat thyroid after 90 days. On the other hand, increases in TSH observed at the 10 mg/kg/day level were sufficient to induce a growth response in the thyroid of the male and female rats after only 14 days of exposure. Thus, substance - induced increases in TSH at levels of ≤ 1.0 mg/kg/day, appeared to be tolerated without consequent alteration of follicular cell morphology (as revealed by standard histopathological findings)

SPERM ANALYSIS AND ESTROUS CYCLICITY:
No statistically significant differences in sperm count, concentration, motiliy or morphology were found at the end of 90 days or following the 30-day recoveryperiod. No statistically significant differences in estrous cyclicity were noted at the end of 90 days or following the 30-day recovery period. The number of females cycling in each group and the mean cycle lengths remained comparable between the control and treatment groups.

MICRONUCLEUS ASSAY
No test article-related changes in bone marrow micronucleus formation of PCE/NCE ratios were observed in males or females after 90 days of treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Analytical verification of doses: analysis of stability samples showed that the substance was stable in reagent-grade water for at least 109 days at concentrations encompassing those used in the 90 -day study. Periodic analysis of animal drinking water solutions demonstrated that they were accurately prepared, with all solutions being within the ± 10 % of the targeted concentrations. No perchlorate was detected in the vehicle control drinking water solutions, and no nitrate was found in any of the solutions analysed, including the RO deionised water used to prepare the test drinking water solutions.

Determination of NOAEL: selection of 1.0 mg/kg/day as the NOAEL requires that the hormonal changes at dosage levels of 1.0 mg/kg/day and lower not to be regarded as adverse effects per se. This approach seems reasonable since: 1) the noted hormonal changes had no effect on the general health of the rats, as measured by their normal growth and the other experimental parameters examines, 2) despite the noted changes in TSH, there were no indications of compensatory thyroid growth at dose levels ≤ 1.0 mg/kg/day and 3) even at the 10 mg/kg/day level, the thyroid growth response was reversible after the 30 -day recovery period. Oral administration of the substance to rats via the drinking water produced thyroid organ weight increases and corresponding histopathological changes in the thyroid after 14 and 90 days at a dosage level of 10 mg/kg/day. These changes were reversible after a nontreatment recovery period of 30 days. Statistically significant changes in TSH and thyroid hormones were observed at dosage levels as low as 0.01 mg/kg/day, however no thyroid organ weight or histopathological effects were observed at dosage levels of ≤ 1.0 mg/kg/day.

Relevance to humans: compared to humans, rat is 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 T3 transport proteins between rats and humans. Both rodents and humans have nonspecific low affinity binding protein, thyroxine-binding globulin, that is virtually undetectable in adult rats. As 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 (appr. 5 to 9 days). It has been estimated that this difference results in a 10 -fold greater requirement for exogenous T4 n the rat with a nonfunctioning thyroid compared to the adult human. Serum T3 levels also show a species difference with half-life in rats being around 6 h, whereas that in humans is about 24 h.

The mean absolute thyroid weights for the 14 and 90 -days interval and 30 -day recovery period are presented in the table below. Values are presented as mean thyroid weight (g)±SD.

Table: Mean absolute Thyroid Weights

	Group 1 0 mg/kg/day	Group 2 0.01 mg/kg/day	Group 3 0.05 mg/kg/day	Group 4 0.2 mg/kg/day	Group 5 1.0 mg/kg/day	Group 6 10.0 mg/kg/day
14-day interval
Males	0.021±0.003	0.021±0.002	0.020±0.004	0.022±0.004	0.021±0.004	0.026*±0.003
Females	0.019±0.003	0.018±0.002	0.019±0.003	0.019±0.003	0.018±0.002	0.020±0.002
90-day interval
Males	0.029±0.005	0.027±0.004	0.027±0.004	0.028±0.004	0.032±0.004	0.038**±0.006
Females	0.022±0.003	0.022±0.004	0.020±0.003	0.020±0.003	0.019±0.004	0.027*±0.005
30-day recovery
Males	0.031±0.005	-	0.031±0.006	-	0.029±0.004	0.032±0.005
Females	0.022±0.004	-	0.022±0.005	-	0.022±0.003	0.024±0.003

* Statistically significant value (p< 0.05)

**Statistically significant value (p<0.01)

The incidence and severity of substance-induced thyroid histopathological effects are presented in the table below. Values reflect the incidence and severity of thyroid follicullar cell hypertrophy with microfollicle formation and colloid depletion. No abnormal thyroid pathology was noted following the 30 -day recovery period.

Table: incidence and severity of substance-induced thyroid histopathological effects

	Group 1 0 mg/kg/day	Group 2 0.01 mg/kg/day	Group 3 0.05 mg/kg/day	Group 4 0.2 mg/kg/day	Group 5 1.0 mg/kg/day	Group 6 10.0 mg/kg/day
14-day interval
Males
Minimal	1/8	0/10	1/10	0/10	0/10	1/10
Mild	0/8	0/10	0/10	0/10	0/10	2/10
Moderate	0/8	0/10	0/10	0/10	0/10	7/10
Total incidence	1/8	0/10	1/10	0/10	0/10	10/10
Females
Minimal	0/10	0/10	0/10	0/10	0/10	4/10
Mild	0/10	0/10	0/10	0/10	0/10	3/10
Total incidence	0/10	0/10	0/10	0/10	0/10	7/10
90-day interval
Males
Minimal	2/10	0/10	0/10	0/10	1/10	1/10
Mild	0/10	0/10	0/10	0/10	0/10	7/10
Total incidence	2/10	0/10	0/10	0/10	1/10	8/10
Females
Minimal	0/10	0/10	0/10	0/10	0/10	6/10
Mild	0/10	0/10	0/10	0/10	0/10	3/10
Total incidence	0/10	0/10	0/10	0/10	0/10	9/10

Applicant's summary and conclusion

Conclusions:

NOAEL (males/female rats) = 1.0 mg/kg/day (nominal)

Executive summary:

The potential effects of the test material when administered to rats in their drinking water over the course of 14 or 90 days were investigated in agreement with EPA test guideline OPPTS 870.3100 . A nontreatment recovery period of 30 days was also included to assess the reversibility of substance-induced effects. Hematology and clinical chemistry parameters, opthalmological examinations, clinical signs, mortality, body weight, histopathology, organ weights were measured. The potential effects on male sperm parameters, females estrous cycling, bone marrow micronucleus formation and serum hormone levels (TSH, T3 and T4) were also investigated.

No toxicologically meaningful differences were observed between the control and treated groups with respect to survival, clinical observations, body weights, food consumpton, opthalmology, hematology, clinical chemistry, estrous cycling, sperm parameters or bone marrow micronucleus formation. Administration of the substance for 90 days did not induce any test article- related gross or microscopic changes in tissues and organs besides the thyroid. Increased absolute and relative thyroid weight was observed at the 10 mg/kg/day group, while decreased T3 and T4 levels, increased TSH levels and thyroid histopathological changes were also noted in the higher dose group. Despite the fact that changes in the hormone levels were also noted in levels below 10 mg/kg/day, there were no indications of a thyroid growth response at dose levels ≤ 1.0 mg/kg/day.

Based on these findings, 1.0 mg/kg/day (nominal) is regarded as the NOAEL (males/female rats).