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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction toxicity data are available for Fe-DTPA from an extended OECD 422 study (with 10 -wk premating period), and for Na-glucoheptonate from a standard OECD 422 study (2 -wk premating).

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test guideline (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See section 13

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

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. In doing so the study became more a one-generation test (OECD 415) 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

Species:

rat

Strain:

Wistar

Sex:

male/female

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

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

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: max 16 days. At the end of the pre-mating period (23 June 2010), each female was caged with one male from the same group. Since the number of females that was mated after 1 week was relatively low in all groups, the mating period was extended initially with another week. Since on Day 12 of the mating period, the number of females that was mated was still relatively low in all groups, males that had not mated during these 12 days were replaced by males of the same dosing group that already had mated with another female. The newly formed pairs were allowed to mate up to another 4 days (1 oestrus cycle).
- 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

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).

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 (parental animals)

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters: not applicable as F1 animals were killed on postnatal day 4.
- Selection of parents from F1 generation when pups were [...] days of age: not applicable as F1 animals were killed on postnatal day 4.
- Age at mating of the mated animals in the study: 15-16 weeks

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-MnA2
- Rationale for animal assignment (if not random): computer randomization proportionately to BW

Parental animals: Observations and 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

Oestrous cyclicity (parental animals):

Not measured

Sperm parameters (parental animals):

Parameters examined:
testis weight, epididymis weight: 12 rats/group
sperm count in epididymides, sperm motility, sperm morphology: 5 rats/group
sperm count in testes, daily sperm production: 5 rats/group

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, because this screening study was ended on day 4 post-partum

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 analysis

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after mating (at least 13 weeks of treatment)
- Maternal animals: All surviving animals at or shortly after day 4 of lactation (almost 14 weeks of treatment)

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

ORGAN WEIGHTS:
- testes, epididymides (12 rats/group)
- adrenals, brain, heart, kidneys, liver, spleen, thymus (5 rats/sex/group)

HISTOPATHOLOGY:
- ovaries, uterus (12 rats/group; control and high dose group
- testes, epididymides, seminal vesicles, prostate, coagulating glands (12 rats/group; control and high dose group
- adrenals, axillary lymph nodes, brain, caecum, colon, femur, Peyer's patches, heart, kidneys, liver, lungs, mesenteric lymph nodes, peripheral nerve, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, urinary bladder (5 rats/sex/group; control and high dose group)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic) externally for gross abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external examinations; pups were stored in a freezer for possible skeletal analyses (not done).

ORGAN WEIGHTS: not done

HISTOPATHOLOGY: not done

SKELETAL ANALYSIS: done

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.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant
females, the number of pregnant females with implants but no pups, females with live pups,
females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost
entirely.
- Pre-coital time (mean number of days), the duration of gestation, the number of corpora
lutea and implantation sites, 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.
- Sperm parameters were evaluated by one-way analysis of variance followed by Dunnett’s
multiple comparison test (epididymal and testicular sperm count and numerical sperm
motility parameters) or by Kruskal-Wallis non parametric analysis of variance and by
Mann-Whitney U test (motility parameters expressed as a percentage and sperm
morphology).

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

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY: Soft faeces were observed in males and females of the high-dose groups in various weeks of the pre-mating period . This finding was most pronounced in the first week of the study. Soft feaces were also observed in some rats of the mid-dose group in the first week of the study only. The findings in the high-dose group occasionally reached the level of statistical significance. The repeated occurrence of soft faeces in the high-dose group was considered treatment-related.
One female of the low-dose group (no. 33) was found dead on day 64 of the pre-mating period. In the fourth week of the study, one female of the mid-dose group (no. 57) showed a hunched body position, piloerection and was thin and weak. On Day 32 of the pre-mating period it was found dead. On Day 3 of the gestation period, a female of the high dose group (no. 85) was killed because it was thin and showed piloerection and abnormal respiration (rales, dyspnoea, decreased frequency). The death of females nos 33 and 85 is likely to have been caused by misdosing. The cause of death of female no. 57 of the mid-dose group could not be examined at necropsy because its organs were already autolytic.

BODY WEIGHT AND FOOD CONSUMPTION: The mean body weight of males of the high-dose group was decreased when compared to the control group, reaching the level of statistical significance as from Day 35 of the study. In addition, the mean body weight change of males of the high-dose group was statistically significantly decreased at various time periods (Days 0-7, 28-49, 63-70). These observed effects on body weight were considered to be related to treatment. Food consumption was not affected by treatment.

TEST SUBSTANCE INTAKE: no effects (gavage)

ESTROUS CYCLE: not examined

SPERM MEASURES: The percentage of motile epididymal sperm cells was statistically significantly decreased in males of the high-dose group. This finding was accompanied by a decrease in the percentage of progressive epididymal sperm cells and an increase in the percentage of static cells. Although the latter two findings did not reach the level of statistical significance, the observed effects were ascribed to treatment. The weight of the cauda epididymides of males of the high-dose group was statistically significantly decreased. In addition, the sperm reserve (sperm count per (3) ml) of males of the high-dose group was statistically significantly decreased. These effects were considered to be related to treatment. No differences were observed on sperm morphology between the control group and the high dose group. No statistical significant differences were found on testicular parenchyma weight, on the number of spermatozoa or on the daily sperm production.

ORGAN WEIGHTS: The relative weights of the kidneys and liver of male and female animals of the high-dose group were statistically significantly increased. These findings were considered treatmentrelated. The absolute weight of the epididymides was decreased in males of the mid- and high-dose
group. The relative weight of this organ was decreased in males of the high-dose group but not in males of the mid-dose group. Therefore, only the decreased weight of the epididymides of males of the high-dose group was considered to be related to treatment.

GROSS PATHOLOGY: no effects

HISTOPATHOLOGY (NON-NEOPLASTIC): no treatment-related effects

HISTOPATHOLOGY (NEOPLASTIC): no changes

HISTORICAL CONTROL DATA: not needed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased relative weight of epididymides, a decrease in sperm motility and epididymal sperm reserve

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:

no effects observed

VIABILITY (OFFSPRING): no effects

CLINICAL SIGNS (OFFSPRING): no effects

BODY WEIGHT (OFFSPRING): no effects

SEXUAL MATURATION (OFFSPRING): not done, pups were necropsied on day 4 post partum

ORGAN WEIGHTS (OFFSPRING): not done

GROSS PATHOLOGY (OFFSPRING): 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.

HISTOPATHOLOGY (OFFSPRING): no effects

OTHER FINDINGS (OFFSPRING): skeletal examinations. 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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects

Reproductive effects observed:

not specified

Conclusions:

Based on the results of this study, viz. a decrease in sperm motility, cauda epididymis weight and in sperm reserve, as observed in male animals treated with the highest concentration of the test substance, the No Observed Adverse Effect Level (NOAEL) for fertility is 500 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 7.8.2.

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.

No treatment-related effects were observed on pre-coital time, mating index, female fecundity index, male and female fertility indices, duration of gestation, number of animals that delivered liveborn pups, number of corpora lutea, number of implantation sites, pre-implantation loss or viability index. Decreased epididymal sperm motility, decreased weight of the cauda epididymides and decreased epididymal sperm reserve were observed in males of the high-dose group. No changes were observed in epididymal sperm morphology, or the weight of testicular parenchyma, the number of spermatozoa per gram testicular parenchyma or on the daily sperm production. The relative weights of the kidneys and liver were increased in both sexes of the high-dose group. The relative weight of the epididymides was decreased in males of the high-dose group. Macroscopic examination at necropsy, microscopic examination of organs and tissues 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, viz. a decrease in sperm motility, cauda epididymis weight and in sperm reserve, as observed in male animals treated with the highest concentration of the test substance, the No Observed Adverse Effect Level (NOAEL) for fertility 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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

The in-life phase of the study was conducted between 05 December 2012 (first day of treatment) and 15 January 2013 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd.
- Age at study initiation: Approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 302 to 346g, the females weighed 191 to 217g.
- Fasting period before study: None.
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food (a pelleted diet was used).
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: Six days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve target values of 21 ± 2°C.
- Humidity (%): Set to achieve target values of 55 ± 15%.
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Distilled water.
Formulations were prepared weekly and stored at 4ºC in the dark.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg of Distilled water.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of Sodium Glucoheptanate.

The concentration of Sodium Glucoheptonate in the test item formulations was determined by direct injection into a mass spectrometer (MS) using an external standard technique.

The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.

Duration of treatment / exposure:

The test item was administered for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Once daily.

Details on study schedule:

Chronological Sequence of Study:
i) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of the pre-pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Remarks:

Doses / Concentrations:
0 (control), 30, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 males and 12 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of a 14-day range-finding study.

Positive control:

None.

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period
and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Water intake was measured daily during the first two weeks of the study.

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)

Sperm parameters (parental animals):

During histopathology, the male testes and epididymides were examined.

Litter observations:

LITTER DATA:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

PHYSICAL DEVELOPMENT:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose grroup.
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix)


HISTOPATHOLOGY:
Samples of the following tissues were removed from five selected males and five selected females from each dose group.
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skin (hind limb)
Eyes
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with brochi)
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina

Tissues were despatched to the histology processing site for processing. Microscopic examination was conducted by the Study Pathologist.

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Reproductive indices:

Mating Performance and Fertility:

The following parameters were calculated from the individual data during the mating period of the parental generation.
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated ÷ Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females ÷ Number of animals mated) x 100

Gestation and Parturition Data :
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring ÷ Number of pregnant females) x 100

Offspring viability indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed for reproduction

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment at dose levels of 30, 300 and 1000 mg/kg bw/day did not produce any convincing toxicological effects of treatment.

Reproductive effects observed:

not specified

Conclusions:

The oral administration to rats of Sodium Glucoheptanate (EC 250-480-2) (incorporating a test item correction factor for 50.5% w/w purity minus 49.5% w/w test item water content) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day did not produce any convincing toxicological effects of treatment and on this basis the ‘No Observed Adverse Effect Level’ (NOAEL) and possible ‘No Observed Effect Level’ (NOEL) for systemic toxicity for either sex was considered to be 1000 mg/kg bw/day.

No treatment-related effects were observed for reproduction, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day (incorporating a correction factor for 50.5% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose

group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross

necropsy examination and histopathological evaluation of selected tissues was performed.

Results:

The oral administration to rats of Sodium Glucoheptanate (EC 250-480-2) (incorporating a test item correction factor for 50.5% w/w purity minus 49.5% w/w test item water content) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day did not produce any convincing toxicological effects of treatment and on this basis the ‘No Observed Adverse Effect Level’ (NOAEL) and possible ‘No Observed Effect Level’ (NOEL) for systemic toxicity for either sex was considered to be 1000 mg/kg bw/day.

No treatment-related effects were observed for reproduction, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well performed GLP studies

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Developmental toxicity data are available for Fe-DTPA from an extended OECD 422 study (with 10 -wk premating period), and for Na-glucoheptonate from a standard OECD 422 study (2 -wk premating).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

The in-life phase of the study was conducted between 05 December 2012 (first day of treatment) and 15 January 2013 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd.
- Age at study initiation: Approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 302 to 346g, the females weighed 191 to 217g.
- Fasting period before study: None.
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food (a pelleted diet was used).
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: Six days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve target values of 21 ± 2°C.
- Humidity (%): Set to achieve target values of 55 ± 15%.
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Distilled water.
Formulations were prepared weekly and stored at 4ºC in the dark.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg of Distilled water.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of Sodium Glucoheptanate.

The concentration of Sodium Glucoheptonate in the test item formulations was determined by direct injection into a mass spectrometer (MS) using an external standard technique.

The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

The test item was administered for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Once daily.

Duration of test:

Up to eight weeks.

Remarks:

Doses / Concentrations:
0 (control), 30, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 males and 12 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of a 14-day range-finding study.

Chronological Sequence of Study:
i) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of the pre-pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Maternal examinations:

The below examinations were performed on maternal females and parental males.

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period
and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Water intake was measured daily during the first two weeks of the study.

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)

PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose grroup.
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix)


HISTOPATHOLOGY:
Samples of the following tissues were removed from five selected males and five selected females from each dose group.
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skin (hind limb)
Eyes
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with brochi)
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina

Tissues were despatched to the histology processing site for processing. Microscopic examination was conducted by the Study Pathologist.

Ovaries and uterine content:

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. The corpora lutea were also counted.

Fetal examinations:

Not examined.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

For reproductive indices see toxicity to reproduction.

Historical control data:

Normal range data for the different parameters examined were used in comparison against the test data.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The oral administration to rats of Sodium Glucoheptanate (EC 250-480-2) (incorporating a test item correction factor for 50.5% w/w purity minus 49.5% w/w test item water content) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day did not produce any convincing toxicological effects of treatment and on this basis the ‘No Observed Adverse Effect Level’ (NOAEL) and possible ‘No Observed Effect Level’ (NOEL) for systemic toxicity for either sex was considered to be 1000 mg/kg bw/day.

No treatment-related effects were observed for reproduction or developmental toxicity within the screening study, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day (incorporating a correction factor for 50.5% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose

group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross

necropsy examination and histopathological evaluation of selected tissues was performed.

Results:

The oral administration to rats of Sodium Glucoheptanate (EC 250-480-2) (incorporating a test item correction factor for 50.5% w/w purity minus 49.5% w/w test item water content) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day did not produce any convincing toxicological effects of treatment and on this basis the ‘No Observed Adverse Effect Level’ (NOAEL) and possible ‘No Observed Effect Level’ (NOEL) for systemic toxicity for either sex was considered to be 1000 mg/kg bw/day.

No treatment-related effects were observed for reproduction or developmental toxicity within the screening study, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See section 13

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

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

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

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

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

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.

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)

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

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.

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well performed GLP studies

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

For Fe-DTPA sperm effects were seen only at a high dose of 1500 mg/kg bw in the presence of other toxic effects, and are considered to be due to zinc deficiency. For Na-glucoheptonate no reproductive effects were seen up to a level of 1000 mg/kg bw. Based on these data, classification for reproductive toxicity is not needed according to GHS. .