Reaction mass of Trisodium tris(D-gluconato(2-)-κO1,κO2)ferrate(3-) and Sodium chloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April-August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted study according to GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

This study was carried out as an extended OECD 422 study in which 12 animals per sex per group were exposed 10 weeks (instead of 2 weeks) prior to mating so that male fertility could be examined and pups evaluated. In doing so the study became more a developmental toxicity test (OECD 414) than a combined subacute/reproscreening test (OECD 422). However, 12 animals/sex/group were used (at least 10 animals/sex/group) to comply to the REACH requirement for Annex VII and VIII studies).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

EC Number:

235-627-0

EC Name:

Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)

Cas Number:

12389-75-2

Molecular formula:

C14-H18-Fe-N3-O10.H.Na

IUPAC Name:

Iron(3+) ion sodium 5-[bis(carboxylatomethyl)amino]-3-{[bis(carboxylatomethyl)amino]methoxy}pentanoate

Details on test material:

Chemical name: Sodium hydrogen [N,N-bis[2-[bis(carboxymethyl)amino]ethyl]glycinato(5-)]ferrate(2-)
Purity: 11.6% (Fe content)
Batch no: CFC 9883
Expiry date: 1 January 2013

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutshland, Sulzfeld, Germany
- Age at study initiation: 5 weeks (females), 6 weeks (males)
- Weight at study initiation: mean weight males ca. 170 g; mean weight females ca. 106 g
- Fasting period before study: not applicable
- Housing: 4 per sex in macrolon cages, with wood shavings as bedding material, and paper strips as environmental enrichment
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 degrees C, reaching a minimum of 19.2 degrees C
- Humidity (%): at least 45% and not exceeding 65%. During several periods, humidity was outside the limits reaching a minimum of 43% and a maximum of 96% during a short period
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 April to 4 August 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Weekly, one bottle of test formulation per dose level was prepared. Preparation of the test formulations was performed one day before the first day of the dosing period and at weekly intervals thereafter until completion of the dosing phase of the study. The different concentrations of the test substance in tap water were prepared by stirring on a magnetic stirrer for at least 1h. The pH of the test formulations of groups 2, 3 and 4 were set between pH 6-7 using sodium carbonate (Na2CO3). Subsequently, under continuous stirring, 8 aliquots (7 days plus 1 extra) were taken according to the volume required for each dosing. Aliqouts were stored in a refrigerator in the dark. On each subsequent day, one aliquot for each group was removed from the refrigerator and allowed to equilibrate to ambient temperature. All aliquots were continuously stirred on a magnetic stirrer during the entire administration period in order to maintain the homogeneity of the test substance in the vehicle.
Sodium carbonate was added to all three test formulations to adjust the acidity of the formulations to pH 6-7: it appeared that the amount of test substance used for the preparation of the test solution for the high-dose group did not dissolve unless the pH was adjusted. On the first 2 days of the study, animals of the low- and mid-dose groups were treated with test formulations without the addition of sodium carbonate. From Day 2 onwards, all animals of the low-, mid- and high-dose groups were treated with test formulations with added sodium carbonate. In week 2 of the study, the pH of the test formulations of the low-, mid- and high-dose groups were marginally higher (resp 7.03, 7.05 and 7.06). This also applied for week 8 of the study, regarding test formulations of the low- and mid-dose groups (resp 7.18 and 7.01).

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

To determine the homogeneity and content of DTPA-FeNaH in gavage liquid, iron was used as a marker for the test item. Iron concentrations in gavage liquid were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES).
The concentrations of iron measured in the gavage liquids prepared on 15 April 2010, 15 June 2010 and 06 July 2010 were ‘close to intended’ (relative difference < 10 %) for all gavage liquids at all dose levels, except for the low-dose level gavage liquids prepared on 15 April 2010 (+10.7%) and the low-, mid- and high-dose level gavage liquids prepared on 06 July 2010 (+10.1%, +11.0% and +13.6%, respectively).

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: max 16 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not done.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

10 weeks pre-mating, 16 days mating, 3 weeks gestation, and 4 days lactation

Frequency of treatment:

single daily application by gavage

Duration of test:

10 weeks pre-mating, 16 days mating, 3 weeks gestation, up to 4 days of lactation

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 500 and 1500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on studies done with EDTA and EDTA-MnNa2
- Rationale for animal assignment (if not random): computer randomization proportionately to BW

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were made once weekly; FOB and motor activity were assessed in week 8 of the pre-mating period.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (males and females) and on day 1 and 4 of lactation (females)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: weekly (at same time as measurement of bw)

WATER CONSUMPTION: No

URINALYSIS: No

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No as females were allowed to litter
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities and skeletal examinations

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, food consumption and organ weights data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- The total number of pups delivered (mean), the mean number of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal-
Wallis nonparametric analysis of variance and by the Mann-Whitney U test.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.
- Skeletal data of the pups was evaluated by the Fisher's exact probability test.

Indices:

- 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

- 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

Historical control data:

Not included.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
soft faeces, increased relative weights of kidneys and liver in animals of the high dose group (see also section 7.5.1)

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No statistically significant differences were found between groups on pup mortality or on sex ratio. The mean number of pups per litter was somewhat lower in all treatment groups than in controls but statistical analysis did not reveal significant differences between groups.
Some abnormalities (subcutaneous heamorrhage, pale, cold and small) were observed occasionally in pups of different groups on lactation Day 1. These abnormalities occurred with the highest incidence in the control group and thus were not ascribed to treatment.
On PN Day 4, the number of thin pups lacking milk in their stomach was statistically significantly increased in the high-dose group when compared to the control group. The 9 pups concerned were from the same litter (dam no. 73). This abnormality is considered to be an indirect effect: the dam lost substantial weight during the lactation period. Statistical analysis on pup body weights on PN Day 1 and 4 and on pup body weight changes (PN Day 1-4) did not reveal significant differences between groups. Stillborn pups of the mid- and high-dose groups and one pup of the high-dose group that died during the lactation period were examined macroscopically. None of the pups showed any remarkable abnormality.
No skeletal malformations or anomalies were found in any of the pups of any groups. Analysis of skeletal variations and retardations did not reveal treatment-related effects. In one pup of each of dam nos 1 (control group), 65 (mid-dose group) and 93 (high-dose group), a hole was observed in the supraoccipital bone.
No statistically significant difference in the incidence of this skeletal varation was observed among the groups. Incomplete ossification of the calcaneus (bilateral) of one pup of dam no. 59 (mid-dose group) was observed. This isolated finding is not ascribed to treatment. Incomplete ossification of cervical bodies was observed in one and three pups of dam nos 1 and 13, respectively, of the control group and in one pup of dam no. 77 of the high-dose group. No statistically significant difference in the incidence of this skeletal retardation was observed. Incomplete ossification of the proximal phalanges of the hindlimbs was observed in allmost all pups of control dams nos 1 and 13 and of high-dose dam no. 73. Statistical analysis showed a decrease in the incidence of incomplete ossification of the proximal phalanges of the hindlimbs in all treatment groups when compared to the control group. This finding is not considered to be related to treatment because the highest incidence of incomplete ossification was found in the control group. It is likely that the (relatively) high incidence of incomplete ossification in allmost all pups of these control dams and high-dose dam, is related to the high number of pups per litter and/or the lower weight of the pups concerned.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no development toxicity observed

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, which did not show any toxicological effects of the test substance on development, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity is ≥1500 mg/kg body weight/day.

Executive summary:

The objective of this study was to provide data on the possible effects of the test substance DTPA-FeNaH on reproductive performance of Wistar rats and the development of pups following daily oral administration at concentrations of 0, 150, 500 or 1500 mg/kg bw of the test substance by gavage to male and female rats during a pre-mating period of 10 weeks, during mating (16 days), and during gestation and lactation until postnatal Day 4 (PN Day 4); see also section 7.5.1 and 7.8.1.

The homogeneity and content of the test substance in the gavage solutions were confirmed by analysis.

Males and females of the high-dose group showed soft faeces in various weeks of the premating period. Daily clinical observations during the gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Mean body weights were decreased in males of the high-dose group from week 5 onwards. There were no treatment-related effects on female body weights during the entire study. No treatment-related effects were observed on food consumption of male and female animals during the entire study.

There were no treatment-related differences in litter size and sex, and pup body weight. Macroscopic examination of the pups at birth and at necropsy, and skeletal analyses of the pups did not reveal any treatment-related changes.

Based on the results of this study, viz. soft faeces (both sexes), decreased body weight gain (males), prolonged prothrombin time (males), increased haemoglobin concentration (males), decreased ALAT activity and chloride concentration (males) and increased relative weights of kidneys and liver (both sexes) as observed in animals treated with the highest concentration of the test substance, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 500 mg/kg body weight/day. Based on the results of this study, which did not show any toxicological effects of the test substance on development, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity is ≥1500 mg/kg body weight/day.