Iodine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-02-09 to 2010-05-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iodine

EC Number:

231-442-4

EC Name:

Iodine

Cas Number:

7553-56-2

Molecular formula:

I2

IUPAC Name:

iodine

Details on test material:

- Name of test material (as cited in study report): Iodine
- Substance type: Pure substance
- Physical state: Solid
- Analytical purity: 99.8%
- Impurities (identity and concentrations): None relevant
- Composition of test material, percentage of components: Iodine pure substance
- Purity test date: 24th November 2009
- Lot/batch No.: Drum 182827052
- Expiration date of the lot/batch: 29 December 2010 (Allocated by testing laboratory)
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:WI (Han)

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. NOTOX BV has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, L'Arbresle Cedex, France
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: Males 329 g ± 20% of the sex mean, Females 192 g ± 20% of the sex mean
- Fasting period before study: Overnight
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm), except for Female 81 which was single housed.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

using a plastic feeding tube

Vehicle:

DMSO

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations of Group 4 and/or a stock solution (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. The Group 2 and 3 formulations were prepared by dilution of the Group 4 formulation or the stock solution. Solutions were stored at ambient temperature.

VEHICLE
- Justification for use and choice of vehicle: Dimethyl sulphoxide (DMSO), specific gravity 1.1 (Merck, Darmstadt, Germany). DMSO was selected based on trial formulations performed at NOTOX.
- Amount of vehicle (if gavage): 1 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight. This dose volume was based on results of a 13 week oral study in the rat in which a NOEL of 1100 mg/kg body weight/day was established (information from MSDS).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (24 March 2010), according to a validated method (NOTOX project 492834). Samples of formulations were analyzed for homogeneity (highest* and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under protection from light and stability over 7 days in a refrigerator was also determined for the highest* and lowest concentrations.

*The highest concentration was 30 mg/mL (analyses were performed before change to 10 mg/mL).

Duration of treatment / exposure:

Males were exposed for 29 days, ie. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Initially, Group 4 animals received 30 mg/kg. However, the dose level was lowered to 10 mg/kg from Day 4 of treatment onwards after one female was euthanized in extremis.

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

0.3 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In order to set the dose levels for the main study, a dose range finding study was performed. Groups of 3 females (11-13 weeks old) were dosed at 1, 10 or 100 mg/kg/day for 10 days by oral gavage. At 1 and 10 mg/kg no toxicologically significant toxicity was noted. Animals treated at 100 mg/kg from Day 2 onwards showed clinical signs consisted of lethargy, hunched posture, piloerection, lean appearance, and/or rales. All three female rats were killed in extremis on Day 4 of treatment.
- Rationale for animal assignment (if not random): by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately after each dosing, once prior to start of treatment and at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, fasted overnight
- How many animals: 5 animals/sex/group
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes, fasted overnight
- How many animals: 5 animals/sex/group
- Parameters checked in table No. 1 were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Selected males were tested during week 4 and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Dose groups that were examined: All groups (5 animals/sex/group)
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- The preserved organs and tissues of the animal that was killed in extremis (no. 80).
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy pups: Group 1 Female 46 and Male 6, and Group 2 Female 56 and Male 16 (both females were non-pregnant).

Other examinations:

Organ Weights: The following organ weights and terminal body weight were recorded from the fllowing animals on the scheduled day of necropsy. Selected 5 animals/sex/qroup: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid; All remaining males: Epididymides, Testes

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
The number of corpora lutea was transformed by using 1/x to obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

One female at 30 mg/kg Group 4, Female no.80) was killed in extremis on Day 2 of the treatment period. This animal showed hunched posture, piloerection, ptosis and pale appearance, and a body weight loss of 10% over one day. At necropsy, many dark red foci on the thymus were noted and reddish discoloration of the gastro-intestinal tract that was also distended with gas. At microscopic examination, this animal had minimal to severe degrees of villous atrophy in the duodenum, jejunum and cecum along with moderate atrophy of the gastric mucosa. These findings indicate a functional disturbance of the gastrointestinal tract which was considered to have contributed to moribundity in this animal. As this animal had been dosed at 30 mg/kg which resulted in a 10% loss of body weight within 24 hours, a relationship to treatment cannot be excluded.

Clinical signs of toxicity were noted at 30 mg/kg for females. No treatment related clinical signs were noted up to 10 mg/kg. At 30 mg/kg, hunched posture, piloerection, pale appearance and ptosis were noted for female no. 80 on the day she was euthanized in extremis (Day 2 of treatment). In addition, Female 75 showed hunched posture on Days 3-5 of treatment. This was considered to have been caused by treatment at 30 mg/kg on Days 1-3 (not from treatment at 10 mg/kg which she received from Day 4 onwards).

Redness and swelling of both ears was noted for female no. 79 (Group 4, 10 mg/kg) over the repro period. Microscopic examination revealed slight unilateral lymphohistiocytic inflammation with cartilage necrosis. At this single incidence it was considered to have occurred by chance and not to be treatment related.
Incidental findings that were noted included scabs and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.

Partly based on these findings, the dose level of Group 4 was lowered on Day 4 of the treatment period from 30 mg/kg to 10 mg/kg.

BODY WEIGHT AND WEIGHT GAIN

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period up to 10 mg/kg.
At 30 mg/kg, body weight loss was noted for five out of ten females (range of 1-12% body weight loss in two days).
Partly based on these findings, the dose level of Group 4 was lowered on Day 4 of the treatment period from 30 mg/kg to 10 mg/kg.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

Food consumption before or after allowance for body weight was similar between treated and control animals. Food consumption was slightly reduced for the first week for females of Group 4 (cages 15-16; not statistically significant). This was considered to have been caused by the high dose level of 30 mg/kg given for the first three days of dosing.


HAEMATOLOGY

No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant reduction in Prothrombin time (PT) and increase in lymphocytes for males at 10 mg/kg (Group 4) were not considered to be toxicologically relevant as they remained within the range considered normal for rats of this age and strain and were considered to have arisen as a result of slightly high or low control values.
Individual increases of neutrophil counts with concurrently reduced lymphocyte counts were noted among the dose groups (both sexes) without a treatment related distribution. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and of no toxicological relevance.

CLINICAL CHEMISTRY

Significantly lower Aspartate aminotransferase (ASAT) values and significantly higher inorganic phosphate values were seen for males at 10 mg/kg (Group 4). Furthermore, significantly lower bile acid counts were noted for males at 3 and 10 mg/kg. Means for these values only just exceeded or remained within the range considered normal for rats of this age and strain.
T4/T3 ratio was increased at 10 mg/kg for the males and at 3 and 10 mg/kg for the females (not statistical significant).
For the females, TSH was reduced for all dose groups however without a dose response relationship and not statistical significant (high variation in control group). Total T3 was reduced for females at 3 mg/kg (not statistical significant) and at 10 mg/kg (p < 0.05), and Total T4 was increased for females at 3 mg/kg (p < 0.05) and at 10 mg/kg (not statistical significant).
For males at 3 mg/kg, an increase in chloride was noted. At 3 mg/kg for females, the statistically significantly lower ASAT and the higher total bilirubin counts occurred in the absence of a treatment-related distribution. Furthermore, the increase in total bilirubin was attributed to a high value noted for animal no. 64. These values were within normal and occurred in the absence of a dose response relationship. Taken together, the changes in chloride, total bilirubin, and ASAT were not considered toxicologically significant.

NEUROBEHAVIOUR

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
One female treated at 0.3 mg/kg (Group 2, no. 54) showed absent grip strength. As it concerning only one animal of the low dose group, it was not considered toxicologically relevant.
The variation in motor activity did not indicate a relation with treatment. Females at 10 mg/kg (Group 4) had higher activity counts (low sensor; not statistically significant). As the values were well within normal limits and it occurred in the absence of any corroborative findings like hyperactivity, the increase was not considered to be toxicologically relevant.

ORGAN WEIGHTS

No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.
At 10 mg/kg (Group 4), the higher liver to body weight ratio seen for males was not reflective of treatment related toxicity because the mean and individual values remained well within the range of data considered normal for this age and strain and no microscopic correlate was noted.
Two females treated at 10 mg/kg (nos. 73 and 75) showed relatively high thymus weights when compared to the concurrent control values. However, these values were still within normal limits and in the absence of microscopic findings, were not regarded toxicologically relevant.
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY

Necropsy did not reveal any toxicologically relevant alterations up to 10 mg/kg.
At 30 mg/kg, the female that was euthanized in extremis (no. 80) was noted with many dark red foci on the thymus and reddish discoloration of the gastro-intestinal tract that was also distended with gas.
Incidental findings included dark red discoloration of the mandibular lymph nodes, black discoloration of the popliteal lymph nodes, an isolated red focus or many dark red foci on the thymus, soft yellow nodule on the tail and body of the left epididymide, thickening of the ears, pelvic dilation of the kidneys, and alopecia over various body regions. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore not considered to be toxicologically relevant.

HISTOPATHOLOGY: NON-NEOPLASTIC

There were no treatment-related microscopic findings.
One female Group 4 animal (30 mg/kg, animal 80) was sacrificed moribund after one day on test. This animal had minimal to severe degrees of villous atrophy in the duodenum, jejunum and cecum along with moderate atrophy of the gastric mucosa. These findings indicate a functional disturbance of the gastrointestinal tract which was considered to have contributed to moribundity in this animal. As this animal had been dosed at 30 mg/kg which resulted in a 10% loss of bodyweight within 24 hours, a relationship to treatment cannot be excluded.
No abnormalities were seen in the reproductive organs of the suspected non-fertile animals (Group 1 female 46 and male 6 and Group 2 female 56 and male 16) which could account for their infertility.
All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain.

Effect levels

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the absence of functional or morphological disturbances supporting the changes noted for clinical biochemistry parameters

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analysis of Dose Preparations

The concentrations analysed in the formulations of Group 2 (0.273 mg/g), Group 3 (2.73 mg/g) and Group 4 (27.3 mg/g) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulations.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the highest concentration level (i.e. 27.3 mg/g) were stable when stored in a refrigerator for at least 7 days. Formulations at the lowest concentration level (i.e. 0.273 mg/g) were stable when stored at room temperature for at least 6 hours, but not stable when stored in a refrigerator for 7 days.

Applicant's summary and conclusion

Conclusions:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in rats by oral gavage following OECD 422. Rats were given 0, 0.3, 3 and 10 mg/kg (initially 30 mg/kg) of Iodine. Based on the absence of functional or morphological disturbances supporting the changes noted for clinical biochemistry parameters under testing conditions, a NOAEL of 10 mg/kg was established after repeated doses of Iodine in Wistar rats.

Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in rats by oral gavage following OECD 422.

 

Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 0.3, 3 and 30 mg/kg of Iodine.

 

After acclimatisation, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to Iodine at 0, 0.3, 3 and 30 mg/kg. Due to severe toxicity at 30 mg/kg, the dose level of Group 4 was adjusted to 10 mg/kg from Day 4 of the study onwards. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

 

The following parameters were evaluated: mortality/viability, clinical signs, functional observations, body weights, food consumption, reproduction/developmental parameters, observations pups, clinical pathology (including thyroid hormones), macroscopy, organ weights, and histopathology. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability.

 

No relevant parental toxicity was observed up to 10 mg/kg. Initially, Group 4 animals received 30 mg/kg. However, the dose level was lowered to 10 mg/kg from Day 4 of treatment onwards after one female was euthanized in extremis. This animal had hunched posture, piloerection, pale appearance and ptosis after one day of dosing. Many dark red foci on the thymus and reddish discoloration of the gastro-intestinal tract that was distended with gas was noted upon macroscopic examination, and minimal to severe degrees of villous atrophy in the duodenum, jejunum and cecum along with moderate atrophy of the gastric mucosa were noted at microscopic examination. These findings indicate a functional disturbance of the gastrointestinal tract which was considered to have contributed to moribundity in this animal. In addition, one female treated at 30 mg/kg showed hunched posture on Days 3-5 of treatment, and body weight loss was noted for five out of ten females treated at 30 mg/kg. Food consumption was slightly reduced for females treated at 30 mg/kg. These findings were considered to have been caused by irritating properties of the test substance as noted during the dose range finding study at 100 mg/kg.

A number of clinical biochemistry changes were noted at 3 and/or 10 mg/kg which included lower aspartate aminotransferase and higher inorganic phosphate levels in blood for males at 10 mg/kg, and lower bile acid counts for males at 3 and 10 mg/kg. Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. Moreover, there were no histopathological correlates that would support these changes. Therefore, these changes were considered not to be of toxicological relevance. The changes noted for thyroid hormone levels were very slight, not statistical significant and/or without a dose response relationship. In addition, as no effects were noted for thyroid weights and histopathology these slight changes were not considered to be toxicologically significant. Up to 10 mg/kg, no treatment-related toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, haematology, macroscopic examination, organ weights, and microscopic examination).

Based on the absence of functional or morphological disturbances supporting the changes noted for clinical biochemistry parameters, a parental NOAEL of 10 mg/kg was established.