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Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23 September 2004 to 10 December 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to characterise the mode of action for test material-related foetotoxicity observed in a previous prenatal developmental toxicity study in rabbits. The specific objectives were to collect time-course data (glutathione and serum iodide) in support of proposed PBPK dosimetry modelling to compare the effects of the test material vs. sodium iodide exposure on prenatal survival and foetal thyroid structure and function and to characterise histopathologic changes in foetal thyroid glands following exposure.
GLP compliance:
yes
Type of method:
in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
Iodomethane
EC Number:
200-819-5
EC Name:
Iodomethane
Cas Number:
74-88-4
Molecular formula:
CH3I
IUPAC Name:
iodomethane
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 months
- Weight at study initiation: 3271 to 4835 g on gestation day 0
- Housing: individually housed in clean, stainless-steel wire-bottom cages suspended above ground corn cob bedding.The bedding was changed at least two
times each week.
- Diet: The feed was offered at gradual increments up to approximately 150 g/animal and then ad libitum throughout the remainder of the study
- Water: ad libitum
- Acclimation period: minimum of 6 days
-Time-mated females were recived on gestation day 14 to 17. The selected females were approximately 6 months old on receipt.

ENVIRONMENTAL CONDITIONS
- Temperature:18.5 to 18.9 °C (65.3 to 65.9 °F)
- Humidity: 46.1 to 56.9 %
- Air changes: Air handling units were set to provide approximately 10 fresh air changes per hour.
- Photoperiod: Light timers were calibrated to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
- Animal exposures were conducted in four 1.5-m^3 and one 1.0-m^3 stainless steel and glass whole-body inhalation chambers. One 1.5-m^3 and one 1.0-m^3 chamber each was dedicated to the control group and three 1.5-m^3 chambers were dedicated to the test material group for the duration of the study. The 1.5-m^3 exposure cage batteries consisted of four individual compartments with approximate dimensions of 17 x 19 x 11 inches. The 1.0-m^3 exposure cage batteries consisted of three individual compartments with approximate dimensions of 22 x 16 x 11 inches. These compartment sizes were acceptable for the 3- or 6-hour exposure period and were consistent with the compartment sizes used in previous studies.
- The chambers were operated under dynamic conditions at slight negative pressure with at least 12 to 15 air changes per hour.
- The does were exposed to the test atmosphere 1 to 4 days (gestation day 23, or gestation day 23 through 24, 25 or 26) for 3 or 6 hours daily. The control group was exposed to clean, filtered air under conditions identical to those used for the groups exposed to test material, except that the control chambers were operated with slight positive pressure.
- Vapours of the test material were generated using a bubbler-type vaporisation system (250-mL gas washing bottle, Ace Glass, Inc., Vineland, New Jersey) containing a 50-mm bottom fritted disc. Regulated compressed air, controlled by a calibrated Cole Parmer rotameter-type flowmeter, flowed to the inlet of the gas-washing bottle containing the test material. The compressed air bubbled through the fritted disc at approximately 10-30 mL/min to create vapours of the test material. The concentrated vapours were directed to the exposure chamber inlet, where the concentration was reduced to the desired level by mixing with the chamber ventilation air.
- Animals were housed in normal animal colony rooms during non-exposure hours. For each daily exposure, the rabbits were transferred from their home cages to transport cages, transported to the exposure room, loaded into exposure chamber caging, exposed for the requisite duration and then returned to their home cages. Food and water were withheld during the exposure periods. The animals were exposed to the test material at approximately the same time each day.
- The comparator material formulation was administered via intravenous infusion, using a calibrated infusion pump, two or four times daily (for approximately 15 minutes per infusion, set to deliver 5 mL/15 minutes) over a 3- or 6-hour period, respectively, on gestation day 23 or during gestation days 23-24, 23-25 or 23-26. Each animal was administered approximately 20.3 μM of sodium iodide per infusion, total approximately 81.2 μM daily. The dose concentration was 0.608 mg/mL sodium iodide; the dosage volume was 5 mL per infusion.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
NOMINAL EXPOSURE CONCENTRATIONS
- A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of test material used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test material used was obtained by weighing the gas-washing bottle containing the test material for each chamber prior to and after each daily exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in litres per minute (LPM) and the exposure duration. The nominal concentration was calculated as follows:
ppm Iodomethane = (Wt. Iodomethane · Mol. Vol. · 10^6) / (MW · Ch. Flow · Exp. Dur.)
Where:
Wt. Iodomethane = weight of test material in grams
Mol. Vol. = Molar volume at 730 mmHg and 21 °C, 25.11 L/mole
10^6 = ppm conversion factor
MW = Iodomethane molecular weight, 141.94 g/mole
Ch. Flow = Daily average chamber flowrate for a given day, in LPM.
Exp. Dur. = Duration of a given day's exposure, in minutes

ACTUAL EXPOSURE CONCENTRATIONS
- Actual exposure concentrations were measured using a gas chromatograph (GC). Samples of the exposure atmospheres from each chamber were automatically collected at approximately 30-minute intervals using a sample loop and a multiposition valve. Samples were collected from the approximate animal-breathing zone of the inhalation exposure chambers.. The following summarises the GC conditions:
Instrument: Hewlett Packard 5890 Series II
Detector: Flame ionisation (FID)
Column: J & W DB-5, 30 m x 0.530 mm I.D., 0.5- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 14 13, Fuel - Hydrogen 17 31, Air 31 290
Temperatures (°C): Injector 250, Column 38, isothermal and Detector 250
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.99 min.
Integrator: Hewlett Packard Model 3396 Series II
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 0, Chart Speed 2.5 cm/min, Peak Area Rejection Value 0, Peak Threshold 0 and Peak Width 0.04
- The chromatograph was standardised using 40-liter Tedlar® gas bags prepared to contain known concentrations of the test material. The standard bags were prepared by injecting known volumes of test material into a 500 mL glass vaporisation bulb set at 78 °C. Compressed air was delivered into the bulb at a known flow rate of approximately 4.0 L/min. to vaporise and dilute the test material and then carry it into the gas bag. The total volume of air was measured by a dry test meter. Concentrations of the gas-phase standards were calculated as follows: Concentration = (VOL · R · T · D · 10^-3 · 10^6) / (L · GMW · P)
Where:
Conc. is in ppm
VOL = volume of test material vaporised into bag in μL
R = universal gas constant, 62.36 L mmHg/mole K
T = nominal laboratory temperature in K (273 + 21 °C = 294 K)
D = density of the test material, 2.280 g/mL
L = volume of air used to prepare bag, 30 L
GMW = gram molecular weight, 141.94 g/mole
P = nominal laboratory barometric pressure, 730 mmHg
10^-3 = μL to mL conversion factor
10^6 = conversion factor to ppm

- Prior to initiation of dosing, the GC was calibrated using a standard curve constructed by a quadratic equation developed from four standards. Each standard was prepared in triplicate prior to the exposure period and analysed with the GC. Prior to exposure on a daily basis, the integrity of the prime calibration curve was checked by analysing one freshly prepared standard. On a rotational basis, a different one of the four standards was used each day. If the analysed concentration was within the acceptable range of the known concentration, the GC was considered within calibration specifications.

DETERMINATION OF HOMOGENEITY OF EXPOSURE ATMOSPHERES
- Evaluation of the homogeneity of exposure concentrations was accomplished during the method development phase of the study prior to animal exposures. Four test locations and a reference location were used for these determinations. The test locations were: right upper front, left lower front, left upper rear and right lower rear, identified as 1, 2, 3 and 4, respectively. Samples were collected as rapidly as possible while always collecting a sample from the reference location and then from one of the four test locations. For each test location, the measured concentration was calculated as a percent difference from the reference location. The homogeneity determination was performed in triplicate for each test material exposure chamber.

AEROSOL MONITORING
- All test material chambers were monitored for aerosol formation during the method development phase of the study. A light scattering aerosol photometer was calibrated and used for monitoring. During the determination, aerosol was not detected at a level equal to or above room air, and it was concluded that no aerosols were present.

RESULTS OF CHARACTERISATION OF EXPOSURE ATMOSPHERES
- NOMINAL EXPOSURE CONCENTRATIONS: The overall mean nominal concentration for each chamber is Group 2 was 29.7 ppm.
- ACTUAL EXPOSURE CONCENTRATIONS: The overall mean analysed concentrations was 20.3 ppm for G2C1, G2C2 and G2C3.
Duration of treatment / exposure:
3 or 6 hours per day
Frequency of treatment:
once daily
Duration of test:
1 to 4 days (gestation day 23, or gestation day 23 through 24, 25 or 26)
Doses / concentrationsopen allclose all
Dose / conc.:
20.3 ppm (analytical)
Remarks:
Test material
Dose / conc.:
81.2 other: µm/day
Remarks:
sodium iodide
No. of animals per sex per dose:
40 females per dose, further divided into 5 females per time point
Control animals:
yes
Statistics:
- Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 and 5 %, comparing each test material-treated and compatator material group to the control group.
- Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, viable foetuses and maternal and foetal glutathione levels were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test material and comparator treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable foetuses, early and late resorptions, total resorptions, pre- and post-implantation loss), external foetal malformations and developmental variations and each particular external malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s was used to compare the test and comparator material-treated groups to the control group.

Results and discussion

Effect levels

Dose descriptor:
other: The study was performed to characterise the mode of action of foetotoxicity
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Results identify iodide as the putative agent responsible for the disruption of the hypothalamic-pituitary-thyroid axis in foetal rabbits
Remarks on result:
not determinable
Remarks:
no NOAEC identified

Observed effects

The study demonstrated that intravenous infusion of sodium iodide administered to pregnant does during GD 23 to 26 could induce identical effects on foetal thyroid structure and function. These results identify iodide as the putative agent responsible for the disruption of the hypothalamic-pituitary-thyroid axis in foetal rabbits.

Any other information on results incl. tables

MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL

- One female in the test material-exposed group was euthanised in extremis on gestation day 28 due to decreased defecation, decreased food consumption and body weight loss; this female had trichobezoar at necropsy. All other females survived to the scheduled necropsies.

- There were no test or comparator material-related clinical findings observed at the daily examinations or 1 hour following exposure; there were no findings midpoint during exposure. Clinical findings, including hair loss or scabbing on various body surfaces, soft stool, decreased defecation, diarrhoea, and brown or yellow material on the anogenital area or base of the tail, were noted prior to or after the exposure period, were observed similarly in the control group and/or were noted in single animals.

 

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS

- During the pre-exposure period (gestation days 17-18 through 22-23), mean body weight gains in the test material-exposed and sodium iodide-treated groups were generally similar to those in the control group. No statistically significant differences were observed. Following the first 2 days of exposure (gestation days 23-24 and 24-25), mean body weight losses were observed in the control, the test material-exposed and/or sodium iodide-treated groups. Mean body weight losses were observed in the test material-exposed or sodium iodide-treated groups compared to a slight gain or slight loss in the control group during gestation days 25-26 and 26-27. When the entire exposure period (gestation days 23-26) was evaluated, a mean body weight loss of 82 g (not statistically significant) was observed in the test material-exposed group compared to the loss of 5 g in the control group. A mean body weight loss (37 g, not statistically significant) was also observed when the entire exposure period (gestation days 23-26) was evaluated in the sodium iodide-treated group. However, mean body weight losses were noted in the test material-exposed and sodium iodide-treated groups during gestation days 27-28 (post-treatment period); the difference in the sodium iodide-treated group was statistically significant (p<0.05) compared to the control group value. When the entire post-treatment period (gestation days 27-29) was evaluated, mean body weight losses of 35 g and 17 g (not statistically significant) were observed in the test material-exposed and sodium iodide-treated groups compared to a gain of 83 g in the control group. During the post-treatment period, only four to five females per group remained for evaluation.

Mean net body weights, net body weight changes and gravid uterine weights in the test material-exposed and sodium iodide-treated groups were similar to the control group at the gestation days 23, 24, 25 and 26 necropsies; no statistically significant differences or trends over time were noted. On gestation day 29, mean gravid uterine weight (384.9 g) in the test material-exposed group was lower (not statistically significant) than the control group value (503.0 g). This reduction correlated with the increase in post-implantation loss observed in this group. Mean net body weights in the the test material-exposed and sodium iodide-treated groups were lower than that in the control group (not statistically significant) and mean net body weight losses were greater than the control group value. The difference was statistically significant (p<0.05) for the mean net body weight loss in the sodium iodide-treated group.

 

MATERNAL FOOD CONSUMPTION

- Mean food consumption, evaluated as g/animal/day and g/kg/day, in the test material-exposed and sodium iodide-treated groups was similar to that in the control group during the pre-exposure period (gestation days 20-21 through 22-23). During the first 2 days of exposure (gestation days 23-24 and 24-25), food consumption in the test material-exposed group was similar to that in the control group. Food consumption in the sodium iodide-treated group was reduced compared to that in the control group during gestation days 23-24 and 24-25; the difference was statistically significant (p<0.05) during gestation days 23-24. During the remainder of the exposure period (gestation days 25-26 and 26-27) through the post-exposure period (gestation days 27-28 and 28-29), food consumption in these groups was reduced (not statistically significant) compared to the control group values. During the post-treatment period, only four to five females per group remained for evaluation.

 

MATERNAL NECROPSY DATA

- One female in the test material-exposed group was euthanised in extremis on gestation day 28 due to decreased food consumption and body weight loss; trichobezoar was observed in the stomach at the time of necropsy. This female also had a distended colon and dark red areas in the duodenum; seven late resorptions were noted in utero. At the schedule necropsies on gestation days 23, 24, 25, 26 or 29, no test or comparator material-related internal findings were observed. Pale liver and/or white areas on the liver were noted in four and two females in the test material-exposed and sodium iodide-treated groups, respectively. These affected females were necropsied on gestation day 23 (two females), 25 (one female) or 29 (three females). Because these findings were not observed in the test material-exposed females in previous studies, they were not attributed to the test or comparator materials. Other macroscopic findings occurred in single animals, are common findings in laboratory rabbits or were observed similarly in the control group.

 

ORGAN WEIGHTS

- Due to the acute nature of the exposure regimen (1 to 4 days), changes in mean organ weights (absolute or relative to final body weight) were not considered test or comparator material-related. The only statistically significant (p<0.05 or p<0.01) differences from the control group were as follows. In the test material-exposed group, mean absolute and relative kidney weights were increased at the gestation day 24 necropsy (6 hours following initiation of daily exposure). A similar increase was not observed in this group following further exposures (at the gestation day 25, 26 or 29 necropsies). Mean absolute and relative brain weights in the test material-exposed group were increased (statistically significant at p<0.05) on gestation day 26 compared to the control group values. Similar increases were not observed on gestation day 29. Mean absolute and relative liver weights in the sodium iodide-treated group were increased (p<0.05 or p<0.01) on gestation day 26 and mean relative liver weight in the test material-exposed group were increased on gestation day 29. On both gestation days 26 and 29, mean absolute liver weights in the control group were lower than those observed in the control group on previous gestation days.

 

LAPAROHYSTERECTOMY DATA

- A test material-related increase in the mean litter proportion of post-implantation loss (53.2 % per litter, four litters evaluated) was observed on gestation day 29 in the test material-exposed group; the difference from the control group value (6.9 % per litter, five litters evaluated) was not statistically significant. This increase was primarily due to an increase in the mean litter proportion of late resorptions (50.4 % per litter) compared to that in the control group (4.7 % per litter). This increase resulted in a decrease in the mean litter proportion of viable foetuses (46.8% per litter) in the the test material-exposed group compared to the control group value (93.1 % per litter). The mean number of viable foetuses (4.0 per dam) in this group was also reduced; the difference was statistically significant (p<0.05) compared to the control group value (7.2 per dam). In the sodium iodide-treated group, the mean litter proportion of post-implantation loss (12.5 % per litter, five litters evaluated) was slightly increased (not statistically significant) compared to the control group value, resulting in a decrease in the mean litter proportion of viable foetuses. However, the increase was due to nearly equal proportions of early and late resorptions. Because early resorption of embryos occurred prior to the initiation of exposure, and because the mean litter proportion of late resorptions (6.8 % per litter) in the sodium iodide-treated group was similar to the control group value (4.7 % per litter), this effect was not attributed to the comparator material. The mean numbers of corpora lutea and implantation sites (gestation day 29) in the sodium iodide-treated group were also increased (statistically significant at p<0.05 or p<0.01) compared to the control group values. However, the formation of corpora lutea and implantation occurred prior to the initiation of treatment; therefore, these increases were not attributed to the comparator material.

- There were no test or comparator material-related effects on intrauterine survival on gestation days 23, 24, 25 or 26. On gestation day 24 (after exposure on that day), the mean litter proportion of postimplantation loss (early resorptions) was increased in the sodium iodide-treated group, resulting in a lower mean litter proportion of viable foetuses. However, the increase in early resorptions was not attributed to the comparator material because treatment did not initiate until gestation day 23, after early resorption of the embryos would have occurred.

 

EXTERNAL FETAL MORPHOLOGICAL DATA

- The numbers of foetuses (litters) available for external morphological evaluation were 340(39), 334(39) and 379(40) in the control, the test material-exposed and sodium iodide-treated groups, respectively. External malformations were observed in 0(0), 1(1) and 2(1) foetuses (litters) in these same respective dose groups and were considered spontaneous in origin. One foetus (gestation day 25) in the test material-exposed group had omphalocele (many loops of intestine protruded through the umbilicus, with remnants of a membranous sac). Two foetuses (gestation day 24) in the sodium iodide-treated group had carpal flexure (unilateral); one foetus also had exencephaly with open eyelids and a body that was shorter than normal.

- There were no external developmental variations. However, blood was noted as thickened and dark in colour for 10(2) foetuses (litters) in the test material-exposed group at the time of blood collection. These findings were not classified as a malformation or developmental variation; the significance was unclear.

 

THYROID/PITUITARY HORMONES

- MATERNAL DATA: In pregnant does, exposure to the test material induced a statistically significant increase in serum TSH concentrations after exposure for 2 days on gestation days 23-24 (135 % of control). A similar increase (136 % of control) was observed after 2 days of exposure to 25 ppm the test material in a previous study. The lack of change after 4 days of exposure and at the other time points in the current study was likely the result of the lower exposure concentration (20 ppm the test material). In the current study, the increase in serum TSH concentrations was not accompanied by alterations in either serum T3 or T4 concentrations. In pregnant does administered sodium iodide by slow bolus infusions, there was a statistically significant increase in serum TSH concentration after exposure for 2 days on gestation days 23-24 (135 % of control). The increase in serum TSH was accompanied by a statistically significant decrease in the serum T3 concentration (77 % of control). The serum T3 concentration was also statistically significantly decreased after exposure for 1 day of sodium iodide administration on gestation days 23 (77 % of control). Generally, no changes were apparent in the overall patterns of maternal TSH, T3, or T4 serum concentrations as the result of increasing duration of sodium iodide administration.

- FOETAL DATA: In foetal serum following maternal the test material exposure, the most frequent alteration in serum hormones was increased TSH concentrations, which was observed after exposure for 3 days on gestation days 23-25 (12-hour recovery, 280 % of control) or for 4 days on gestation days 23-26 (no recovery, 268 % of control; and 72-hour recovery, 400 % of control). In contrast, the serum TSH concentration was statistically significantly decreased after exposure on gestation days 23 (18-hour recovery samples only, 67 % of control). There was a statistically significant decrease in serum T4 concentrations after exposure to the test material on gestation days 23-25 (6-hour recovery, 30% of control). The increase in foetal serum TSH concentration in the current study was consistent with results from a previous study in which thyroid and pituitary hormone serum concentrations were measured after 2 days (gestation days 23-24) or 4 days (gestation days 23-26) of exposure to 0 or 25 ppm the test material. In the foetuses of does administered sodium iodide, the overall pattern of effects was similar to that observed for the test material. The most common alteration was increased serum TSH concentrations, which was observed after exposure on gestation days 23-25 (6-hour recovery, 206 % of control; and 12-hour recovery, 253 % of control), and gestation days 23-26 (no recovery, 274 % of control). Alterations in thyroid serum hormone levels included a statistically significant decrease in serum T3 concentrations after exposure to sodium iodide on gestation days 23-24 (no recovery, 73 % of the test material) and a statistically significant decrease in serum T4 concentrations after exposure to sodium iodide on gestation days 23-25 (6-hour recovery, 5 % of control). Overall, the increase in serum TSH concentration was not accompanied by concordant changes in the profiles for the serum T3 or T4 concentrations.

 

SERUM IODIDE

- MATERNAL DATA: Maternal serum iodide concentrations were substantially increased in the test material-exposed and sodium iodide-treated groups and increased with the duration of maternal exposure. Maternal serum iodide concentrations in the test material-exposed group were approximately twice the serum iodide concentrations in the sodium iodide-treated group during (i.e., 3-hour time point) and immediately following (i.e., 6-hour time point) the daily exposure periods.

- FOETAL DATA: Foetal serum iodide concentrations were substantially increased in the test material-exposed and sodium iodide-treated groups as early as 3 hours into maternal exposure and increased with the duration of maternal exposure. Concentrations of iodide in foetal serum following the daily maternal exposure on gestation day 26 (6 hours) were 2- to 3-fold higher than concentrations following exposure on gestation day 23 (6 hours) in both the test material- and sodium iodide-treated groups. Mean foetal serum iodide concentrations were generally 2- to 3-fold higher than maternal concentrations in both the test material-exposed and sodium iodide-treated groups; baseline levels of iodide in the control animals were also higher in foetal versus maternal serum. Foetal serum iodide concentrations in the test material-exposed group were generally about twice as high as serum iodide concentrations in the sodium iodide-treated group.

 

HEMOGLOBIN ADDUCT

- Concentrations of S-methylcysteine adducts were measured in maternal and foetal haemoglobin as a marker of the test material exposure.

- MATERNAL DATA: Concentrations of S-methylcysteine adducts in maternal haemoglobin were slightly elevated in the test material-exposed group compared to the control group. S-methylcysteine was detected in the haemoglobin of control does (air exposure) at an average concentration of 69.7 ± 9.3 nmol/g haemoglobin. In does exposed to the test material, the mean S-methylcysteine concentration was increased to 93.4 ± 5.0 nmol/g haemoglobin.

- FOETAL DATA: Concentrations of S-methylcysteine adducts in foetal haemoglobin were slightly elevated in the test material-exposed group compared to the control group, suggesting that some unreacted the test material is transferred to the foetus. The mean concentration in control foetal haemoglobin was 86.9 ± 20.1 nmol/g. In foetal haemoglobin from does exposed to the test material, the concentration averaged 131.1 ± 11.0 nmol/g.

 

GLUTATHIONE

- MATERNAL DATA: Concentrations of glutathione were measured in maternal liver, blood and nasal respiratory epithelium. Mean glutathione concentration in the liver of the test material-exposed females was lower (statistically significant, p<0.01) than the control group value following 6 hours of exposure on gestation day 23; however, the concentrations were similar to those in the control group prior to and following exposure on gestation day 24 (0-hour and 6-hour evaluations). Mean concentration in maternal blood was lower (statistically significant, p<0.01) than the control group value following the 6-hour exposure on gestation day 24, but not on gestation day 23. No other statistically significant differences from the control group were noted in blood or liver. There were also no declines in mean glutathione concentrations in nasal respiratory epithelium at any of the time points evaluated.

- FOETAL DATA: Concentrations of glutathione were measured in foetal liver and blood. Mean concentrations in foetal blood were lower (statistically significant, p<0.05) than the control group values following 6 hours of the test material exposure on both gestation days 23 and 24; mean glutathione concentration at the 0-hour evaluation on gestation day 24 was similar to the control group value. No statistically significant differences from the control group were noted in foetal liver.

 

MICROSCOPIC EXAMINATION

- MATERNAL: Microscopic findings in the thyroid glands of does (five per group) euthanised on gestation day 26 were generally considered to be within normal limits; however, two animals exposed to the test material displayed very minimal follicular cell hypertrophy and colloid depletion. A third doe exposed to the test material also displayed very minimal depletion of colloid. Very minimal depletion of colloid was also thought to be present in one animal given sodium iodide intravenously. It should be emphasised that thyroid findings in all does could not be clearly distinguished from normal biologic variation.

- FOETAL: Thyroid follicular hypertrophy and colloid depletion were present in the thyroid glands of foetuses exposed to the test material and treated with sodium iodide; following 2, 3 or 4 days of maternal exposure (gestation days 24 through 26 evaluations), 60 to 100 % of the foetuses were affected. These findings were not observed in any control group foetuses, nor in any the test material-exposed or sodium iodide-treated foetuses on gestation day 23. Hypertrophy was characterised by minimally to mildly increased volumes of foamy to finely vacuolated cytoplasm within follicular cells, while colloid depletion was typified by minimally to moderately reduced accumulations of colloid within follicular cavities. For gestation days 24, 25 and 26, the incidence and severity of these findings were similar between groups exposed to the test material or treated with sodium iodide. At gestation day 29 following 3 days of recovery, the incidences of follicular cell hypertrophy and colloid depletion were slightly higher in the test material-treated group; however, the severity was noticeably greater in foetuses exposed to the test material. The following table presents the incidence of microscopic findings in the test material-exposed (Group 2) and sodium iodide-treated (Group 3) foetuses (all control group foetuses had no remarkable observations).

Summary of selected foetal data

Dose (ppm)

Iodomethane

 

Sodium Iodide

Day of Laparohysterectomy

24

25

26

29

 

24

25

26

29

NO. OF FOETUSES

EXAMINED

 

20

20

20

 

20

20

20

16

 

Thyroid

 

 

 

 

 

 

 

 

 

Hypertrophy

16

20

16

6

 

12

18

18

5

M-

16

20

9

2

 

12

18

8

5

M+

0

0

7

4

 

0

0

10

0

 

Colloid depletion

 

1

 

5

 

13

 

6

 

 

0

 

9

 

17

 

6

M-

1

4

6

1

 

0

5

7

6

M+

0

1

1

2

 

0

4

4

0

M++

0

0

6

3

 

0

0

6

0

M- minimal; M+ mild; M++ moderate

Summary of selected maternal and foetal clinical chemistry data

Parameter ± SD

[foetal data]

Group / Maternal Exposure

 

TSH

(ng/dL)

T3

(ng/dL)

T4

(μg/dL)

 

1. None (GD 23, 6 hour)

 

0.5±0.10     [1.2±0.4]

 

180±23.8     [4.5±2.55]

 

1.76±0.27   [0.07±0.03]

2. Iodomethane (GD 23, 6 hour)

0.52±0.19   [1.1±0.2]

173±12.3     [6.5±4.77]

1.75±0.40   [0.10±0.05]

3. Sodium iodide (GD 23, 6 hour)

0.78±0.58   [1.1±0.3]

139±5.2       [9.0±4.61]

1.26±0.38   [0.13±0.09]

 

1. None (GD 24, 6 hour)

 

0.46±0.11   [1.9±0.5]

 

173±16.4     [10.4±2.01]

1.43±0.42   [0.05±0.04]

2. Iodomethane (GD 24, 6 hour)

0.62±0.04*  [1.7±0.4]

158±19.7     [13.6±4.63]

1.44±0.46   [0.08±0.05]

3. Sodium iodide (GD 24, 6 hour)

0.62±0.08*  [2.0±0.6]

133±22.3*    [7.6±1.98]

1.15±0.71   [0.03±0.03]

 

1. None (GD 25, 12 hour)

 

0.56±0.05   [1.7±0.4]

 

160±36.3  [13.1±6.50]

 

1.33±0.24   [0.20±0.11]

2. Iodomethane (GD 25, 12 hour)

0.68±0.20   [2.7±0.6]

136±33.6   [13.3±5.76]

0.95±0.85   [0.06±0.09*]

3. Sodium iodide (GD 25, 12 hour)

0.72±0.04   [3.5±1.5*]

127±30.7   [10.8±6.04]

0.38±0.39   [0.01±0.01*]

 

1. None (GD 25, 18 hour)

 

-                 [1.5±0.2]

 

-                 [12.0±2.44]

 

-                 [0.08±0.07]

2. Iodomethane (GD 25, 18 hour)

-                 [4.2±1.1*]

-                 [13.2±5.61]

-                 [0.00±0.00]

3. Sodium iodide (GD 25, 18 hour)

-                 [3.8±1.1*]

-                 [14.5±4.15]

-                 [0.01±0.02]

 

1. None (GD 26, 6 hour)

 

0.58±0.24   [1.9±0.9]

 

122±24.25    [15.4±3.09]

 

0.60±0.38    [0.06±0.04]

2. Iodomethane (GD 26, 6 hour)

0.58±0.15   [5.1±1.5*]

114±25.0   [26.6±12.62]

0.84±0.89    [0.03±0.05]

3. Sodium iodide (GD 26, 6 hour)

0.60±0.07   [5.2±2.9*]

123±15.5   [15.7±2.39]

0.61±0.66    [0.01±0.01]

 

1. None (GD 29)

 

0.56±0.11   [1.1±0.2]

 

168±29.7   [23.9±4.85]

 

0.77±0.35    [0.14±0.04]

2. Iodomethane (GD 29)

1.05±0.65   [4.4±3.4*]

150±18.2  [49.4±30.17]

0.40±0.36    [0.10±0.15]

3. Sodium iodide (GD 29)

0.74±0.33   [1.7±0.5]

149±44.4   [31.8±9.10]

1.03±0.67    [0.09±0.05]

* Statistically (p<0.01) significant

Summary of maternal and foetal serum iodide data

Parameter ± SD

[fold increase from control]

Group / Maternal Exposure (GD)

 

 

GD and sample time

Maternal iodide

(ng/dL)

Foetal iodide

(ng/dL)

 

1. None

 

(GD 23, 3 hour)

6.89±2.28

114±14

2. Iodomethane GD 23

(GD 23, 3 hour)

7500±488 [1089]

15100±4620 [132]

3. Iodomethane GD 23

(GD 23, 3 hour)

3400±346 [493]

7420±1330 [65]

 

1. None

 

(GD 23, 6 hour)

 

48.6±56.6

 

179±77

2. Iodomethane GD 23

(GD 23, 6 hour)

9570±4750 [197]

27800±9250 [155]

3. Iodomethane GD 23

(GD 23, 6 hour)

5830±611 [120]

14900±8120 [83]

 

1. None

 

(GD 24, 6 hour)

 

23.5±13.4

 

155±24

2. Iodomethane GD 23 - 24

(GD 24, 6 hour)

14300±2360 [609]

33200±11900 [216]

3. Iodomethane GD 23 - 24

(GD 24, 6 hour)

6290±528 [268]

23000±6320 [149]

 

1. None

 

(GD 25, 12 hour)

 

14.3±6.4

 

161±16

2. Iodomethane GD 23 - 25

(GD 25, 12 hour)

5110±1760 [357]

40100±15700 [249]

3. Iodomethane GD 23 - 25

(GD 25, 12 hour)

4330±1610 [303]

25000±8530 [155]

 

1. None

 

(GD 25, 18 hour)

 

19.7±11.4

 

217±55

2. Iodomethane GD 23 - 25

(GD 25, 18 hour)

4470±3250 [227]

32000±128500 [147]

3. Iodomethane GD 23 - 25

(GD 25, 18 hour)

1740±563 [88]

18600±4900 [86]

 

1. None

 

(GD 25, 6 hour)

 

10.5±7.0

 

171±66

2. Iodomethane GD 23 - 26

(GD 25, 6 hour)

16600±6800 [1581]

72600±23200 [425]

3. Iodomethane GD 23 - 26

(GD 25, 6 hour)

9630±11000 [917]

35900±17200 [210]

Applicant's summary and conclusion

Conclusions:
The increase in methylcysteine adduct concentration in foetal haemoglobin indicates that some unreacted test material may be delivered directly to the foetus, the weight of evidence from these exposure data, when considered collectively with the timing of key developmental events in the rabbit foetal thyroid gland during gestation days 23-26, indicates that high concentrations of iodide in foetal rabbits leading to disruption of the hypothalamic-pituitary-thyroid axis is the mode of action for the late stage foetal deaths following maternal exposure to test material on gestation days 23 to 26.
Executive summary:

The objective of the study was to characterise the mode of action for test material-related foetotoxicity observed in a previous prenatal developmental toxicity study in rabbits. The study was performed under GLP conditions.

The specific objectives were to collect time-course data (glutathione and serum iodide) in support of proposed PBPK dosimetry modelling, to compare the effects of test material versus sodium iodide exposure on prenatal survival and foetal thyroid structure and function and to characterize histopathologic changes in foetal thyroid glands following exposure. Kinetic markers of exposure, including haemoglobin adducts, were measured for dosimetry purposes. Activity of 5'-deiodinase was measured in maternal and foetal brain, liver, kidney and placental tissues (reported separately).

One group of 40 time-mated female New Zealand White rabbits was exposed by whole-body inhalation to the test material, test material, for 3 or 6 hours daily. The target concentration was 20 ppm. Actual mean concentration was 20.3 ppm. A concurrent control group, composed of 40 females, was exposed to filtered air. The females in the control and test material groups were exposed on gestation days 23, 24, 25 and/or 26. A comparator group composed of 40 females received 81.2 μM sodium iodide in sterile water for injection, USP, via four, 15-minute intravenous infusions (20.3 μM per infusion) 2 hours apart over a 6-hour period or via two, 15-minute infusions 2 hours apart over a 3-hour period on the same gestation days. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. Rabbits were euthanized in sets of five at specific time points during the exposure schedule. A laparohysterectomy was performed on each female on the appropriate gestation day. The uteri, placentae and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The foetuses were examined for external malformations and developmental variations.

Following maternal exposure to test material on gestation days 23-26, the mean litter proportion of late foetal resorptions was increased (approximately 10-fold, not statistically significant due to small sample sizes and variability among litters) on gestation day 29 (50.4 % per litter) compared to the control group (4.7% per litter). The study design that utilised five rabbits per time point did not provide sufficient statistical power to characterise this difference as statistically significant, given the variability of the endpoint. The lack of statistical significance is not an issue in this study, given that this effect has been well documented in previous studies. The mean litter proportions of late foetal resorptions on gestation days 23, 24, 25 and 26 in the test material-exposed group were similar to control group values. The increased number of late foetal resorptions on gestation day 29 correlated with a decrease in the mean litter proportion of viable foetuses (46.8 % per litter) in the test material-exposed group compared to the control group (93.1 % per litter). Mean gravid uterine weight on gestation day 29 was also lower (not statistically significant) in the test material-exposed group (384.9 g) compared to the control group value (503.0 g); the decrease was attributed to the increase in postimplantation loss in this group. These findings were consistent with those observed in a previous study. In the sodium iodide-exposed group, the mean litter proportions of late foetal resorptions, viable foetuses and mean gravid uterine weight were similar to control group values. There were no test or comparator material-related effects on foetal weights. Maternal and foetal serum iodide concentrations were increased in the test material (inhalation) and sodium iodide (intravenous infusion) groups and increased with the duration of maternal exposure. After 3 hours of exposure to 20 ppm test material on gestation day 23, the maternal serum iodide level increased compared to the control group level of 7 ng/mL to 7500 ng/mL, and after 6 hours the level increased to 9570 ng/mL. After 3 hours of exposure to 20 ppm test material on gestation day 23, foetal serum iodide increased from the control group level of 114 ng/mL to 15100 ng/mL, and after 6 hours of exposure, increased dramatically to 27800 ng/mL. The increase in maternal and foetal serum iodide levels in rabbits exposed to test material for 3 hours indicated that test material is rapidly metabolised, releasing iodide into circulation. The further increase in foetal serum iodide measured after a full 6 hours of exposure suggested that a mechanism exists for iodide accumulation in the rabbit foetus. Mean maternal serum iodide concentration in the test material-exposed group was approximately 197-fold higher than control serum iodide concentrations on gestation day 23 (after 1 day of exposure) and approximately 1581-fold higher on gestation day 26 (after 4 days of exposure). Foetal serum iodide concentrations were 2- to 3-fold higher than maternal concentrations in both the test material and sodium iodide exposed groups. This suggested that iodide preferentially accumulates in the rabbit foetuses. Maternal and foetal serum iodide concentrations in the test material group were approximately twice the serum iodide concentrations in the sodium iodide group. Concentrations of S-methylcysteine adducts in maternal and foetal haemoglobin were slightly elevated in the test material-exposed group compared to the control group, suggesting that some test material may have been distributed to the foetus. S-methylcysteine was detected in the haemoglobin of control does (air exposure) at an average concentration of 69.7 ± 9.3 nmol/g haemoglobin. The mean concentration in control foetal haemoglobin was 86.9 ± 20.1 nmol/g. In does exposed to test material, the mean S-methylcysteine concentration was increased to 93.4 ± 5.0 nmol/g haemoglobin. In foetal haemoglobin from does exposed to test material, the concentration averaged 131.1 ± 11.0 nmol/g. Test material-related decreases in mean glutathione concentrations were noted following 6 hours of exposure in the test material-exposed group maternal liver (gestation day 23) and blood (gestation day 24) and foetal blood (gestation days 23 and 24). The differences from the control group were statistically significant. Mean concentrations in the maternal nasal respiratory epithelium and foetal liver in the test material-exposed group were similar to those in the control group. The decreases in glutathione following test material exposure suggested that test material is metabolized in the maternal liver and perhaps maternal and foetal blood. Thyroid follicular cell hypertrophy and colloid depletion were present in the thyroid glands of foetuses exposed to both test material and sodium iodide, beginning on gestation day 24. These findings were not observed in any control group foetuses. On gestation days 24, 25 and 26, the incidence and severity of these foetal thyroid findings were similar between these two exposure groups and increased with the duration of maternal exposure to test material or sodium iodide. On gestation day 29 (following 3 days of recovery), the incidence of follicular cell hypertrophy and colloid depletion was similar between the test material and sodium iodide groups; however, the severity remained greater in foetuses exposed to test material. Microscopic findings in the thyroid glands of does euthanized at gestation day 26 were generally considered to be within normal limits and could not be clearly distinguished from normal biologic variation. One female in the test material-exposed group was euthanized on gestation day 28 due to decreased food consumption and body weight loss; this female had trichobezoar at necropsy. All other females survived to the scheduled necropsy. Mean maternal body weight losses and reduced body weights and food consumption (occasionally statistically significant) were observed in the test material-exposed and sodium iodide treated groups during the last 2 days of the exposure/treatment period. These reductions continued into the post-treatment period. There were no test or comparator material-related clinical or macroscopic findings or effects on organ weights and foetal morphology.

The window of foetal susceptibility to test material exposure was previously identified to occur from gestation days 23 to 26. A previous baseline study conducted in rabbits showed that this window corresponds directly with the timing of potentially critical developmental events in the rabbit foetal thyroid gland, including the appearance of colloid in the follicular lumen and significant increases in T4 and T3 production beginning on gestation day 22. Results of the current study confirmed findings from those previous studies, showing an test material-related increase in the mean litter proportion of late foetal deaths, increased maternal and foetal serum iodide concentrations, and microscopic changes in the foetal thyroid, including colloid depletion, hypertrophy of the follicular epithelium and vacuolation of the epithelial cytoplasm. Furthermore, the study demonstrated that intravenous infusion of sodium iodide administered to pregnant does during gestation days 23 to 26 could induce identical effects on foetal thyroid structure and function. These results identify iodide as the putative agent responsible for the disruption of the hypothalamic-pituitary-thyroid axis in foetal rabbits. The mean litter proportion of late foetal deaths was not increased in the sodium iodide group compared to the control group; however, this is likely due to the much lower dose of iodide administered to the maternal rabbits in the sodium iodide group, resulting in an internal dose of iodide in maternal and foetal serum that was about one-half that achieved by the 20 ppm test material exposure. Test material exposure and sodium iodide administration caused statistically significant increases in serum TSH concentrations in foetal serum samples. No consistent alterations in foetal serum T3 and T4 or maternal serum TSH, T3 and T4 concentrations were observed. The foetal TSH results were consistent with thyroid hormone measurements reported in previously in which rabbit does were exposed to 0 or 25 ppm test material for 2 days on gestation days 23-24 or 4 days on gestation days 23-26. Although the increase in methylcysteine adduct concentration in foetal haemoglobin indicates that some unreacted test material may be delivered directly to the foetus, the weight of evidence from these exposure data, when considered collectively with the timing of key developmental events in the rabbit foetal thyroid gland during gestation days 23-26, indicates that high concentrations of iodide in foetal rabbits leading to disruption of the hypothalamic-pituitary-thyroid axis is the mode of action for the late stage foetal deaths following maternal exposure to test material on gestation days 23 to 26.

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