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Toxicological information

Toxicity to reproduction

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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
Study period:
16 August 2010 - 06 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Horst, the Netherlands.
- Age at study initiation: Approximately 11 weeks. These animals will be slightly older than stated in the OECD 408 guideline; however this excess with only two weeks will not have any influence on the study integrity.
- Weight at study initiation: males 335-338 mean, females 235-240 mean.
- Housing:
Pre-mating: Animals will be housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females will be caged together with males on a one-to-one-basis in Macrolon plastic cages (Mill type, height 18 cm).
Post-mating: Males will be housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage.
Females will be individually housed in Macrolon plastic cages (Mill type, height 18 cm).
Lactation: Pups will be kept with the dam until termination in Macrolon plastic cages (Mill type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) will be supplied. Certificates of analysis are examined and then retained in the NOTOX archives. During activity monitoring, animals are housed individually in Macrolon plastic cages (Mill type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment will be provided during activity monitoring
- Diet (e.g. ad libitum):Free access to pelleted rodent diet (SM RlM-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants are examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) are examined and archived
- Acclimation period:At least 5 days prior to start of treatment.

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

IN-LIFE DATES: 16 August 2010 - 10 December 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance will be administered undiluted and the dose volume (ml/kg body weight) will be calculated as follows: Dose level (g/kg) I purity (%) and density (g/cm3)
Group 1 animals will receive the same dose volume as animals of Group 4.
Actual dose volumes will be calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females will be individually housed in Macrolon plastic cages (Mill type, height 18 cm).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The males and females will be exposed for at least 78 days prior to mating, during mating, and up to the day prior to necropsy (at least 90 days of dosing). Females will not be dosed during littering. Pups will not be treated directly, but will be potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals will be dosed up to the day prior to scheduled necropsy.

Details on study schedule:
Not applicable, only F0 mated.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw/day
Basis:
other: Adjustment will be made for purity (35%) and density (1.2653) of the test substance.
No. of animals per sex per dose:
10
Control animals:
other: yes, concurrent water
Details on study design:
- Dose selection rationale: Dose levels were selected based on. results of a 10-day dose range finding study (NOTOX Project 494987) in which no toxicity was seen at 1000 and 2000 mg/kg.
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
- Section schedule rationale (if not random): no data
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become more severe, will be sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7). The time of death will be recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, detailed clinical observations will be made in all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals this will also be performed outside the home cage in a standard arena. Arena observations
will not be performed when the animals are mating, or housed individually.
The time of onset, degree and duration will be recorded. All symptoms will be recorded and graded according to fixed scales:
Maximum grade 1: grade 0 =absent, grade 1 =present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females will be weighed on the first day of exposure and weekly thereafter. Mated females will be weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. In order to monitor the health status animals may be weighed more often. This will be documented in the study raw data.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females will be measured on Days 0, 4,7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, of all males of the control and high dose group, additional slides of the testes will be prepared to examine staging of spermatogenesis.
Litter observations:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter will be determined. If possible, defects or cause of death will be evaluated. Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become
more severe, will be sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7).

Clinical signs: At least once daily, detailed clinical observations will be made in all animals.

Body weights: Live pups will be weighed on Days 1 and 4 of lactation

Sex: Sex will be determined for all pups on Days 1 and 4 of lactation.

GROSS EXAMINATION OF DEAD PUPS: Yes, Pups surviving to planned termination will be killed by decapitation between Days 5-7 of lactation. All pups will be sexed and descriptions of all external abnormalities will be recorded. The stomach will be examined for the presence of milk. If possible, defects or cause of death will be evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy will be conducted on the following days:
Condition/Day of necropsy
Females which deliver/Lactation Days 5-7
Females which fail to deliver3/Post-coitum Days 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Females with total litter loss4/Within 24 hours of litter loss.
Males/ Following completion of the mating period (a minimum of 90 days of dose administration).
Spontaneous deaths1/As soon as possible after death and always within 24 hours.
Euthanized in extremis1/When pain, distress or discomfort is considered not transient in nature or is likely to become more severe.

All animals will be subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities will be recorded. The number of former implantation sites and corpora lutea will be recorded for all paired females.

Identification marks: not processed
Adrenal glands
Aorta
Brain - cerebellum, mid-brain, cortex
Caecum
Cervix
Clitoral gland
Colon
Coagulation gland
Duodenum
Epididymides 2
Eyes (with optic nerve (if detectable) and Harderian
gland) 2
Female mammary gland area
Femur including joint
Heart
Ileum
Jejunum
Kidneys
(Lacrimal gland, exorbital)
(Larynx)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
Preputial gland
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes 2
Thymus
Thyroid including parathyroid if detectable
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions

1 Recognizable fetuses will be examined externally, sexed (if possible), and euthanized by decapitation (if necessary).
2 Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and MiIIi-Ro water (Millipore Corporation, Bedford, USA» and transferred to formalin after fixation for at least 24 hours.
3 In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique (Ref. 1) in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt,
Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA».

Tissues/organs mentioned in parentheses need only be examined by the pathologist if changes in macroscopic appearance are indicative of (potential) toxicity.

The following slides will be examined by a pathologist:
The preserved organs and tissues of all animals/sex of Groups 1 and 4.
The additional slides of the testes of all males of Groups 1 and 4 to examine staging of spermatogenesis.
The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
All gross lesions of all animals (all dose groups).
The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups (this will be notified by protocol amendment).
The mammary gland area of all females with total litter loss in which no pups of the litter have milk visible in the stomach (this will be notified by protocol amendment).

On detection of possible treatment-related changes in the organs of any animal in the high dose group the histological examination will be extended to that particular organ of the animals of Groups 2 and 3 (males and/or females). All abnormalities will be described and included in the report. An attempt will be made to correlate gross observations with microscopic findings

Postmortem examinations (offspring):
See litter observations.
Statistics:
The following statistical methods will be used to analyse the data:
If the variables can be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate will be applied for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-to-one rank test) will be applied if the data can not be assumed to follow a normal distribution. The Fisher Exact-test will be applied to frequency data. All tests will be two-sided and in all cases p < 0.05 will be accepted as the lowest level of significance.
Group means will be calculated for continuous data and medians will be calculated for discrete data (scores) in the summary tables. Test statistics will be calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values. Additional methods of statistical analysis may be used. The methods and results will be described in the report.
Reproductive indices:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups or pups/number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100
Offspring viability indices:
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day n-m= (number of pup surviving m days/number of liveborn on day n) x100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male fetuses or pups on day n/ number of live fetuses or pups on day n) x 100

Results and discussion

Results: P0 (first parental animals)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

For details see "700-459-3, Repeated dose toxicity: oral-extended OECD 422, Van Otterdijk, 2011, RS"

Reproduction data
No effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Gestation
Duration of gestation was similar between treated and control animals. The gestation index showed no dose-related trend among the dose groups.

Parturition/Maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on fertility were observed in the study.

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)

Mortality
One pup of the control group, and one pup each at 500 and 200 mg/kg were found dead during the first day of lactation. The mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Clinical signs
Incidental clinical symptoms of pups consisted of a wound in the neck, a blue spot on the neck, scabbing of the neck, pale/cold appearance, small size, absence of milk in the stomach, cold. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age.

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

Macroscopy
The pup found dead at 2000 mg/kg had no milk in the stomach. Incidental macroscopic findings among surviving pups included scabbing on the neck, small size, scabbing of the whole body, and scabs on the head. The nature and incidence of these findings remained within the range considered normal for pups of this age.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with Complexation products of sodium tartrate with iron trichloride by oral gavage in male and female Wistar Han rats at dose levels of 500, 1000 and 2000 mg/kg revealed parental toxicity at 1000 and 2000 mg/kg. Findings representing local toxicity consisted of inflammatory/hyperplastic lesions in the large intestines at 1000 and 2000 mg/kg along with presumed secondary white blood cell changes. Findings indicative of systemic toxicity included higher kidney, liver and spleen weight, and clinical biochemistry changes at 2000 mg/kg. No reproduction and developmental toxicity was observed for treatment up to 2000 mg/kg body weight/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental local NOAEL: 500 mg/kg
Parental systemic NOAEL: 1000 mg/kg
Reproduction NOAEL: at least 2000 mg/kg
Developmental NOAEL: at least 2000 mg/kg
Executive summary:

Title

Combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Complexation products of sodium tartrate with iron trichloride in rats by oral gavage.

Methods

Guidelines

The study was based on the following guidelines:

1) OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996.

2) OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

3) OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

4) OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

5) EC No 440/2008 B.26: Sub-chronic Oral Toxicity Test: Repeated dose 90-day toxicity study in rodents, May 2008.

6) OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, September 1998.

7) OPPTS 870.3100, 90-Day Oral Toxicity in Rodents, August 1998.

Rationale for dose levels

Based on the results of a 10-day dose range finding study (NOTOX Project 494987), the dose levels for this combined 90-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 500, 1000 and 2000 mg/kg.

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 500, 1000 and 2000 mg/kg. Males were exposed for 90 or 91 days during pre-mating and mating. The females were exposed during 104-109 days during pre-mating, mating, post-coitum, and at least 4 days of lactation.

Evaluated parameters

The following observations and examinations were evaluated: mortality/viability, clinical signs (daily), functional observations, locomotor activity, body weight (weekly), food consumption (weekly), reproduction/developmental parameters, observations pups, ophthalmoscopic examination (during pretest and Week 13), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues.

Results/discussion

Parental results

Treatment up to 2000 mg/kg was well tolerated by the animals. No statistically significant changes were noted during functional observation tests, and at body weight and food consumption

measurements.

At 500, 1000 and 2000 mg/kg dark coloured faeces was observed in both sexes during treatment. In females, at necropsy, these findings correlated to black intestinal contents of primarily the large intestines. In males, however, macroscopy did not reveal such a finding. These in-life/necropsy findings were considered to be due to staining properties of the test substance (a dark green liquid). In males and females at 1000 and 2000 mg/kg, increased incidences and severities above background level of neutrophilic infiltrates, acute inflammation, goblet/epithelial cell hyperplasia, foci of brown pigment and/or edema in the large intestines were found. These findings appeared slightly more pronounced in males than in females. The higher relative eosinophil counts in both sexes at 2000 mg/kg as well as the slightly higher relative neutrophil counts, higher white blood cell counts and lower lymphocyte counts in males at 2000 mg/kg may be attributed to these effects in the large intestine. At 2000 mg/kg, a notably higher kidney weight was recorded for males and females (38% and 31% higher than controls, respectively). At 500 and 1000 mg/kg, kidney weights were 10-15% higher than controls. Although the higher urea levels at 2000 mg/kg and to a lesser extent at 1000 mg/kg in both sexes as well as lower sodium and chloride levels in males at 2000 mg/kg would suggest an effect on kidney function, there were no treatment-related histopathological correlates that were indicative of kidney lesions. However, the magnitude of kidney weight increase at 2000 mg/kg was considered to be adverse in nature. At 500 and 1000 mg/kg, the magnitude of effects was clearly less, and in combination with absence of morphological correlates not to be adverse in nature. The slightly higher alanine aminotransferase activity and higher bile acid level in males and/or females at 1000 and/or 2000 mg/kg may be related to the slightly higher liver weight at 2000 mg/kg. However, there were no histopathological lesions indicative of altered liver function. Hence, these changes were considered not to be adverse in nature.

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (2000 mg/kg). No changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

Developmental results

No developmental toxicity was observed up to the highest dose level tested (2000 mg/kg). No changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Conclusion

Treatment with Complexation products of sodium tartrate with iron trichloride by oral gavage in male and female Wistar Han rats at dose levels of 500, 1000 and 2000 mg/kg revealed parental toxicity at 1000 and 2000 mg/kg. Findings representing local toxicity consisted of inflammatory/hyperplastic lesions in the large intestines at 1000 and 2000 mg/kg along with presumed secondary white blood cell changes. Findings indicative of systemic toxicity included higher kidney, liver and spleen weight, and clinical biochemistry changes at 2000 mg/kg. No reproduction and developmental toxicity was observed for treatment up to 2000 mg/kg body weight/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental local NOAEL: 500 mg/kg

Parental systemic NOAEL: 1000 mg/kg

Reproduction NOAEL: at least 2000 mg/kg

Developmental NOAEL: at least 2000 mg/kg