Copper iodide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted as per OECD guideline, following GLPs

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations

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 (100%)
- Purity test date: 24th November 2009
- Lot/batch No.: Drum 182827052
- Expiration date of the lot/batch: 20 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:Wl (Han)

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: 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

Vehicle:

DMSO

Details on 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).

Details on mating procedure:

- M/F ratio per cage: one female/one male
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
- Any other deviations from standard protocol: Mating of female no. 50 was overlooked, since live offspring was delivered by this animal.

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

Details on study schedule:

- Age at mating of the mated animals in the study: 13 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested
Animals were dose 30 mg/kg bw/day for 4 days because of clinical signs, dose was lowered to 10 mg/kg bw/day

Doses / concentrations 2

Remarks:

Doses / Concentrations:
3 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.3 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/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:

No

Examinations

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight and epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups (day 1 and 4 of lactation), viability (daily), body weights (day 1 and 4 of lactation), clinical signs (daily).
Necropsy: Pups surviving to planned termination were killed by decapitation on lactation Day 5, 6 or 7. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals at lactation Days 5-7; Females which were non pregnant (No 46 and No 56) at post-coitum Day 26 (females with evidence of mating)

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed Table 2.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed on lactation Day 5, 6 or 7.
- These animals were subjected to postmortem examinations macroscopic as follows:

GROSS NECROPSY
- Gross necropsy consisted of external aexaminations. The stomach was examined for the presence of milk

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.

Reproductive indices:

Mating (%): Number of females mated/Number of females paired x 100
Fertility index (%): Number of pregnant females/Number of females paired x 100
Conception index (%): Number of pregnant females/Number of females mated x 100
Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the biginning of parturition

Offspring viability indices:

Percentage live males at first litter check: Number of live male pups at first litter check/Number of live pups at first litter check x 100
Percentage live females at first litter check: Number of live female pups at first litter check/Number of live pups at first litter check x 100
Percentage of postnatal loss days 0-4 of lactation: Number of dead pups on day 4 of lactation/Number of live pups at first litter check x 100
Viability index (%): Number of live pups on day 4 of lactation/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

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 FOOD CONSUMPTION (PARENTAL ANIMALS)

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.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

Not examined (gavage study)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception index, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
The statistically significantly increased number of implantation sites noted for low dose females (0.3 mg/kg) was not considered toxicologically relevant, as no dose response was seen and values were within normal limits.


ORGAN WEIGHTS (PARENTAL ANIMALS)

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 (PARENTAL ANIMALS)

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 (PARENTAL ANIMALS)

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 (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects were noted.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)

The gestation index and duration of gestation were similar between controls and all treated groups. Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment

CLINICAL SIGNS (OFFSPRING)

Incidental clinical symptoms of pups consisted of small size, scabbing of the abdomen and pale appearance were noted. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING)

Body weights of pups were considered to have been unaffected by treatment.

SEXUAL MATURATION (OFFSPRING)

Not examined

ORGAN WEIGHTS (OFFSPRING)

Not examined

GROSS PATHOLOGY (OFFSPRING)

The incidental macroscopic finding of small size was noted for one pup. The nature and incidence of these findings remained within the range considered normal for pups of this age, and was therefore not considered to be toxicologically relevant.

HISTOPATHOLOGY (OFFSPRING)

Not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects were noted.

Overall reproductive toxicity

Reproductive 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:

Based on the results, a reproduction and developmental No Observed Adverse Effect Level (NOAEL) of 10 mg/kg was derived

Executive summary:

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

The study was based following guidelines from Organisation of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (1996) and The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2000).

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.

After acclimatisation, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance 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 in the parental animals: 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. Pups were examined for viability, clinical signs, and body weights were determined. All pups were sexed and descriptions of all external abnormalities were recorded at necropsy. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability.

No reproduction/developmental toxicity was observed at any dose level. Based on these results, a reproduction and developmental No Observed Adverse Effect Level. (NOAEL) of 10 mg/kg was derived.

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.