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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined repeated dose toxicity study with the reproductive/ developmental toxicity screening test
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The following experimental and recording deficiencies can be reported for the publication: test substance is a mixture, therefore, it is unclear, if the observed effects were due to the iron content of the mixture alone; mixture has a low pH (3.7), which might cause the observed effects in the intestine; exposure duration was not clearly described; exposure duration to long; clinical signs were not fully described; detailed clinical examinations were not fully described; historical control data and individual data missing.
Justification for type of information:
see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996 - 03 - 22
Deviations:
yes
Remarks:
prolonged exposure during pre-mating phase as described according to guideline at least 14 days premating exposure in the study 12 weeks premating exposure
Principles of method if other than guideline:
The OECD guideline 408 (1998) was also followed during the conducted of study.
GLP compliance:
yes
Limit test:
no
Justification for study design:
not applicable

Test material

Constituent 1
Chemical structure
Reference substance name:
Iron trichloride
EC Number:
231-729-4
EC Name:
Iron trichloride
Cas Number:
7705-08-0
Molecular formula:
FeCl3
IUPAC Name:
iron(3+) trichloride
Constituent 2
Chemical structure
Reference substance name:
disodium;(2S,3S)-2,3-dihydroxybutanedioate
Cas Number:
21106-15-0
Molecular formula:
C4H4Na2O6
IUPAC Name:
disodium;(2S,3S)-2,3-dihydroxybutanedioate
Test material form:
liquid
Details on test material:
- State of aggregation: liquid
- specific gravity: 1.2653 g/mL
- pH: 3.7
- colour: dark green

On a component basis, the test item is composed of about 4% sodium tartrate, approximately 10% mesotartaric acid, approximately 7% chloride, approximately 4% iron, approximately 7% sodium, approximately 0.3% sodium oxalate and approximately 65% water.
In the publication it is stated that analytical data show that FemTA comprises 3.7% iron, the maximum use level of FemTA in salt is 324 ppm (12 ppm × 100% = 1200/3.7% [iron content in 100% of product] = 324 ppm) or 0.0324%.
Specific details on test material used for the study:
- Source: Akzo Nobel Industrial Chemicals, Inc. (Amersfoort, The Netherlands)

- Stability and storage: the product was in complete aqueous solution and did not tend to settle out or precipitate. It was stated to be stable through the time frame of the study, and stored at room temperature in a sealed tin foil-covered container protected from light.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan (Horst, the Netherlands)
- Females non-pregnant: yes
- Age at study initiation: 11 weeks
- Weight at study initiation: males 307 - 351g; females 223 - 251g
- Housing: housed in Macrolon plastic cages (18 cm height, Bio-Services, Uden, the Netherlands) bedding material: sterilized sawdust (Litalabo SPPS, Argenteuill, France) and paper (Enviro-dri, Wonham Mill Ltd., Surrey, United Kingdom) as cage enrichment.
- Acclimatization and premating periods: rats were housed in groups of 5 in Macrolon Type MIV cages
- Cohabitation period (mating): 1 male / 1 female of the same group were housed together in Makrolon Type MIII cages
- Post mating: males were housed as in the premating phase while females were individually housed in their respective Makrolon Type MIII cages.
- During locomotor activity monitoring: all animals were individually housed in Hi-temp polycarbonate cages (Ancare Corp., Bellmore, N.Y., U.S.A.) without cage enrichment or bedding material.

- Diet (ad libitum): pelleted diet SM R/M (SSNIFF Spezialdiaeten GmbH, Soest, Germany)
- Water (ad libitum): municipal tap water

- Acclimatization period: length of period not stated, but acclimatization was done

ENVIRONMENTAL CONDITIONS
- Temperature: 17.6°C to 22.8 ◦C
- Humidity: 31% to 81%
- Air changes: approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Remarks:
distilled
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
FemTA was used as supplied

Dosing was achieved by the applied volume. Dosing volume (mL/kg bw) was calculated based on: (dose level [g/kg]/density [g/cm3]×100/35 [purity adjustment]) where the latest body weight measurements were used. Controls received the same dosing volume (distilled water) as the high-dose group.
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female (same treatment group avoid sibling mating

Once mating was detected the, the respective males and females were separated. This day was designated day 0 postcoitum
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- males: 12 weeks pre-mating, during mating, and up to the day prior to scheduled sacrifice during the post-mating period (total of 90/91 days)
- females (pregnant/littered): 12 weeks pre-mating, during mating, during pregnancy (gestation) and during at least 4 d of lactation (ranging from a total period of 104 to 109 days)
- females (mated but not pregnant): 12 weeks pre-mating, during mating, during the post-mating period (ranging from a total period of 104 to 109 days)
Frequency of treatment:
once daily, 7 days per week
Details on study schedule:
not specified
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
equivalent to 20 mg/kg bw/d Fe(III)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
equivalent to 40 mg/kg bw/d Fe(III)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
equivalent to 80 mg/kg bw/d Fe(III)
No. of animals per sex per dose:
treatment groups 10 male + 10 female
control group 10 male + 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a preliminary 10-d dosing range finding study, no evidence of toxicity was noted at doses of up to 2000 mg/kg bw/d.
Positive control:
not specified

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: - clinical signs at least once daily immediately after dosing
- mortality/viability at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly intervals outside of the home cage in a standard arena.
Any abnormal findings were recorded with respect to symptom and graded according to a fixed scale.

BODY WEIGHT: Yes
- Time schedule:
males: first day of treatment and weekly thereafter
females: first day of treatment and weekly thereafter gestation on days 0, 4, 7, 11, 14, 17, and 20 and during lactation days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule: weekly except during the mating phase. Following mating, food consumption by females was measured on the same days as body weight measurements were recorded.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: not specified

WATER CONSUMPTION: Yes
- Time schedule for examination: subjective evaluation only

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimation period and week 13
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: isoflurane (Abbott BV, Hooffddrop, the Netherlands) in nitrous oxide/oxygen (Air Products, Amsterdam, the Netherlands) anaesthesia.
- Animals fasted: Yes, overnight
- How many animals: 8-10 per group
- Parameters checked: activated partial thromboplastin time, APTT; haematocrit, HCT; mean corpuscular haemoglobin, MCH; mean corpuscular haemoglobin concentration, MCHC; mean corpuscular volume, MCV; mean platelet volume, MPV; platelet; PT, prothrombin time, PLT; red blood cell count, RBC; reticulocyte, RET; red blood cell count, RBC; red cell distribution width, RDW; white blood cells, WBC; neutrophil (%WBC); lymphocytes (%WBC); monocytes (%WBC); eosinophiles (%WBC); basophiles (%WBC); haemoglobin

CLINICAL CHEMISTRY: Yes;
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: isoflurane (Abbott BV, Hooffddrop, the Netherlands) in nitrous oxide/oxygen (Air Products, Amsterdam, the Netherlands) anaesthesia.
- Animals fasted: Yes, overnight
- How many animals: all males and 9-10 females per group
- Parameters checked: alanine aminotransferase, ALAT; aspartate aminotransferase, ASAT; alkaline phosphatase, ALP; total protein; albumin; total bilirubin; urea; creatinine; glucose; cholesterol; bile acids; sodium; potassium; chloride; calcium; inorganic phosphate

PLASMA/SERUM HORMONES/LIPIDS: not specified

URINALYSIS: not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observations:
- Time schedule: males during week 13; females near the end of the lactation period
- Dose groups that were examined: all animals
- Function tested: hearing ability, papillary reflex, static righting reflex, and grip strength.

Locomotor activity:
- Time schedule: males during week 12; females near the end of the lactation period
- Dose groups that were examined: all animals
- Function tested: all animals were caged individually and monitored under normal light conditions for activity by a computerized monitoring system (Kinder Scientific LLC, Poway, Calif., U.S.A.). Total movements and ambulations were recorded. Ambulations represent movements characterized by a relocation of the entire body position, such as walking. Total movements represent all movements made by the animals, including ambulations, but also smaller or finer movements like grooming, weaving, or movements of the head.

IMMUNOLOGY: not specified
Oestrous cyclicity (parental animals):
not specified
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight
Additional slides of the testes were prepared from the control- and high-dose group to examine the staging of spermatogenesis (not further specified).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not specified

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

- Number of live and dead pups on day 1 of lactation and daily thereafter
- Clinical signs determined at least once daily by visual inspection.
- Body weights of live pups determined on days 1 and 4 of lactation.
- Sex determined at days 1 and 4 of lactation,
- percentage of live males and females
- percent postnatal loss from days 0 to 4 of lactation.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All animals were fasted overnight and sacrificed Next, the animals were subject to macroscopic evaluation. The numbers of former implantation sites and corpora lutea were recorded. Furthermore, the following organs weights were recorded adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus, prostate, seminal vesicles, and thyroid gland

HISTOPATHOLOGY: Yes
The following organs and tissues were fixed in 10% buffered formalin and preserved at necropsy from all vehicle control- and high-dose rats:
adrenal glands, aorta, brain, cecum, colon, cervix, clitoral gland, coagulation gland, duodenum, female mammary area, femur, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular and mesenteric), oesophagus, ovaries, pancreas, pituitary gland, Peyer’s patches, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, salivary gland, skeletal muscle, skin, tongue, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid and parathyroid, trachea, urinary bladder, uterus, and vagina, as well as all gross lesions.

Additional slides were prepared from the cecum, colon, and rectum of low- and mid-dose groups to assess a potential treatment-related effect. The samples were embedded in paraffin wax, sectioned (2 to 4 μm), and stained with haematoxylin and eosin (Klinipath).
Postmortem examinations (offspring):
SACRIFICE/ GROSS NECROPSY
- The F1 offspring surviving to 5 to 7 d postbirth were killed by decapitation. Pups were sexed and any external abnormalities recorded. The stomach was examined for the presence of milk. Where possible, defects or cause of death were determined or evaluated.
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett test (Dunnett 1955) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Steel test was applied if the data could not be assumed to follow a normal distribution. The Fisher’s exact test was applied to frequency data. All tests were 2-sided and in all cases P < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances.
Reproductive indices:
Mating index (%) = (females mated/females paired) × 100
Fertility index (%) = (pregnant females/females paired) × 100
Conception index (%) = (pregnant females/females mated) × 100
Gestation index (%) = (females with living pups on day 1/pregnant females) × 100
Duration of gestation
Offspring viability indices:
Viability index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Following statistically significant changes were considered treatment-related.
1) Males:
- 2000 mg/kg bw/d: higher white blood cell (WBC, P<0.01) counts, higher relative neutrophil counts (P<0.05) and lower relative lymphocytes counts (P<0.05) compared to control were noted.

2) Females:
- 2000 mg/kg bw/d: higher relative eosinophil counts (P<0.05) compared to control were observed.

Please also refer to the field “Attached background material”.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Compared to control, following statistically significant changes were considered treatment-related.
1) Males:
- 1000 and 2000 mg/kg bw/d: increased blood urea nitrogen (BUN, P<0.01) and bile acid levels (P<0.01)
- 2000 mg/kg bw/d: increased alanine aminotransferase (ALAT, P<0.01), reduced sodium (P<0.05) and chloride (P<0.01) concentrations

2) Females:
- 2000 mg/kg bw/d: increased blood urea nitrogen (BUN, P<0.05) and reduced chloride (P<0.05) concentrations.

Please also refer to the field “Attached background material”.
Endocrine findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Compared to control, following statistically significant changes were considered treatment-related.
Males and Females:
- 1000 and 2000 mg/kg bw/d: increase in both incidence and severity of neutrophilic infiltrates, acute inflammation, goblet/ epithelial cell hyperplasia, foci of brown pigment and/or oedema in the rectum, colon and cecum was observed.
Please also refer to the field “Attached background material”.
Histopathological findings: neoplastic:
not specified

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS:
1) Males and Females:
- 500, 1000 and 2000 mg/kg bw/d: the test item appeared to be well tolerated. Dark coloured faeces were observed from week 2 of the reproduction phase onwards, week 7 of the premating phase onwards, and week 5 on the pre-mating phase onwards, respectively. This was due to the staining properties of the material (FemTA). Salivation was also noted shortly after dosing. This was likely a response to the taste of the substance rather than due to a systemic reaction.

MORTALITY
1) Males and Females:
- 500, 1000 and 2000 mg/kg bw/d: no mortality/morbidity were noted in any of the animals of both sex throughout the experimental test period
2) Females:
- 0 mg/kg bw/d: One female was killed in extremis on day 70 of the premating period. No cause of death could be determined.

BODY WEIGHT AND WEIGHT CHANGES
1) Males:
- 500 mg/kg bw/d: body weights and body weight gain of males were similar to control levels
- 1000 and 2000 mg/kg bw/d: from week 8 and 3 onwards respectively, achieving a level of statistical significance (P<0.01).
2) Females:
- 500 mg/kg bw/d: body weights and body weight gain of females were similar to control levels
- 1000 mg/kg bw/d: statistically significant (P<0.05) lower body weight gain on day 4 of the lactation phase.
- 2000 mg/kg bw/d: statistically significant (P<0.05) lower body weight gain in week 5 of the premating phase
All of the changes were of small magnitude (not exceeding 10%) and were considered of minimal toxicological significance. Please also refer to the field “Attached background material”

FOOD CONSUMPTION
1) Males and Females:
- 500, 1000, and 2000 mg/kg bw/d: No statistically significant changes in food consumption were recorded at any time during the study at any dose level compared to the control.

WATER CONSUPTION
1) Males and Females:
- 500, 1000, and 2000 mg/kg bw/d: Based on a subjective evaluation, there were no discernible differences between the treated groups and controls with respect to water consumption.

OPHTHALMOLOGICAL FINDING
1) Males and Females:
- 500, 1000 and 2000 mg/kg bw/d: The ophthalmological examinations (data not shown) revealed no effect of FemTA treatment.

HAEMATOLOGICAL FINDINGS
1) Males
- 1000 and 2000 mg/kg bw/d: higher mean corpuscular haemoglobin of males at 1000 (P<0.05) and 2000 mg/kg (P<0.01) occurred in the absence of concurrent changes in red blood cell parameters. Also, the means were within the range considered normal for rats of this age and strain.
- 2000 mg/kg bw/d: lower red blood cell counts (P<0.05) and lower platelet (PT) counts (P<0.05) were considered to be within normal ranges for rats of this age and strain. It was not considered of biological importance since the opposite effect (that is, an increase in PT) would be expected in case of target organ toxicity.
2) Females
- 500, 1000 and 2000 mg/kg bw/d: with increasing dose a tendency to decreasing lymphocyte and increasing neutrophil counts was noted in treated females.
3) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: compared to the controls, the results showed no statistical significant changes for: activated partial thromboplastin time, APTT; haematocrit, HCT; mean corpuscular haemoglobin concentration, MCHC; mean corpuscular volume, MCV; mean platelet volume, MPV; prothrombin time, PLT; reticulocyte, RET; red cell distribution width, RDW; monocytes (%WBC); basophiles (%WBC); haemoglobin
Please also refer to the field “Attached background material”.

CLINICAL BIOCHEMESTRY FINDING
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: compared to the controls, the results showed no statistical significant changes for: aspartate aminotransferase, ASAT; alkaline phosphatase, ALP; total protein; albumin; total bilirubin; creatinine; glucose; cholesterol; potassium; calcium; inorganic phosphate
Please also refer to the field “Attached background material”.

BEHAVIOUR (FUNCTIONAL FINDINGS)
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: Hearing ability, pupillary reflex, static righting reflex, and grip strength were similar to the control group in all treated animals. In the motor activity assessment all groups showed a similar habituation profile with high activity in the first testing interval that decreased over the duration of the test period (data not shown). In high-dose females, lower total movement and, in all treated females, lower ambulation counts, were considered to have occurred due to slightly lower total movements/ambulation counts halfway through the measurement period (that is, during the 6th and 7th 5-min interval of the twelve 5-min intervals measured). Since this was of a temporary nature it was considered not to represent a change of toxicological significance.

ORGAN WIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
1) Males
- 1000 mg/kg bw/d: relative testes weights (P<0.05) were increased compared to control.
- 2000 mg/kg bw/d: relative spleen weights (P<0.01) were increased. Changes occurred in the absence of clear dose–response relationships and with respect to the findings in 2000 mg/kg bw/d dose males, were largely attributable to lower terminal body weights as evidenced by the lack of any statistically significant effects on absolute organ weight of the spleen.
2) Females
- 500 mg/kg bw/d: absolute thyroid weight (P<0.05) was reduced
- 1000 mg/kg bw/d: brain weights (P<0.05) were increased. Changes occurred in the absence of clear dose–response relationships.
3) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: the results showed no statistical significant changes for compared to control for the following organs: adrenal glands, epididymites, heart ovaries, thymus, uterus, prostate and seminal vesicles.
Please also refer to the field “Attached background material”.

GROSS PATHOLOGICAL FINDINGS
1) Females
- 500, 1000 and 2000 mg/kg bw/d: at necropsy black contents in the gastrointestinal tract, primarily the cecum and colon, were observed in most treated females, but not in males of any dose group.

2) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: all other findings were within the normal range of variation for rats of this age and strain.

HISTOPATHOLOGICAL FINDINGS (NON-NEOPLASTIC)
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: the remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain and included, for example, grey–white foci in the lungs, tan foci on the preputial glands, reduced size of preputial glands or testes/epididymites, pelvic dilatation of the kidneys, and enlarged lymph node. None of these lesions were related to treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES
1) Males
- 500, 1000 and 2000 mg/kg bw/d: normal testes weight and normal epididymites weights. Staging of spermatogenesis in the testes did not provide any evidence of test article-related impairment of the spermatogenetic cycle. The investigated parameters were not further specified.

REPRODUCTIVE PERFORMANCE
1) Males
- 500, 1000 and 2000 mg/kg bw/d: There were no treatment-related morphological findings in the reproductive organs, including the testes/epididymites, prostate gland, seminal vesicles preputial gland, ovaries, uterus, and vagina. There were no statistically or biologically significant changes in any of the parameters measured. mating index, fertility index, conception index, gestation index and duration of gestation

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
(Reproductive toxicity)
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
(general toxicity)
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
(equivalent to 20 mg/kg bw/d Fe(III))
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

CLINICAL SIGNS
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: visual inspection of the F1 animals from days 0 to 4 of lactation were inconspicuous.

MORTALITY / VIABLITY
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: There were no statistically or biologically significant changes in any of the parameters measured. percentage of live F1 males and F1 females, percent postnatal loss from days 0 to 4 of lactation and viability index (F1) compared to the control.

GROSS PATHOLOGICAL FINDINGS
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d:
There was no effect of treatment on the results of the external macroscopic examination, like external abnormalities and presence of milk in the stomach.

OTHER EFFECTS
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: the sex ratio of the offspring were normal.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Lynch et al. 2013 administered to groups of 10 male and 10 female Harlan Wistar rats an iron trichloride containing complexation/reaction product, termed FemTA by oral gavage. FemTA is a mixture of sodium tartrate [D(–)- and L(+)-tartaric acid and mesotartaric acid], sodium hydroxide, and iron trichloride. The composition of the product was approximately 4% sodium tartrate, 10% mesotartaric acid, 7% chloride, 4% iron, 7% sodium, 0.3% sodium oxalate, and 65% water. FemTA was administered to the groups at dose levels of 500, 1000, and 2000 mg/kg body weight/d (equivalent to 20, 40, or 80 mg of iron/kg body weight/d). Male rats were dosed prior to and during mating and up to the day prior to scheduled sacrifice during the post-mating period (total of 90/91 days).The females were treated with the substance prior to mating and during mating as well as during gestation and lactation (at least up to lactation day 4) (total of 104 to 109 d).. During the treatment period the substance was administered once daily, 7 days per week. A control group was run concurrently.

During the observation of the parental (P) animals, no test item-related effects were observed in animals for clinical signs, mortality, body weight and weight changes, food consumption, water consumption, ophthalmological findings, behaviour (functional findings), and gross pathology.,

However, compared to the vehicle control, treatment-related effects were observed in parental (P) rats receiving the substance at dose levels of 1000, and 2000 mg/kg body weight/d.. During the haematological examination, an increase in white blood cell count (p < 0.01) and relative neutrophil counts (p < 0.05) as well as a decrease in relative lymphocyte count (p < 0.05) were noted for male rats of the 2000 mg/kg bw/day dose level. Furthermore, an increase in relative eosinophil counts (p < 0.05) were observed in females at the 2000 mg/kg bw/day dose level. Also, treatment-related effects were observed for clinical biochemical findings. At the 1000 and 2000 mg/kg bw/day dose levels, increased blood urea nitrogen (p < 0.01) and bile acid levels (p < 0.01) were noted for male rats. Furthermore, an increase in alanine aminotransferase (p < 0.01) as well as a decrease in sodium and chloride concentrations were observed in male rats at the 2000 mg/kg bw/day dose level. Increased blood urea nitrogen (p < 0.05) and decreased chloride concentrations (p< 0.05) were recorded for female rats at the 2000 mg/kg bw/day dose level.

At the 500, 1000 and 2000 mg/kg bw/day dose levels, an increased absolute (p < 0.05) and relative kidney weight (p< 0.01) was observed in the male rats of the parental generation. In addition, an increased absolute (p < 0.05) and relative liver weight (p < 0.01) was recorded for male rats of the 2000 mg/kg bw/day dose level. At the 1000 and 2000 mg/kg bw/day dose levels, elevated organ weights were observed for absolute (2000 mg/kg bw/day dose level only; p < 0.01) and relative kidney weights (p < 0.01) for female rats of the parental generation. Alterations in kidney weight were clearly treatment-related, and, given the magnitude, were considered adverse at the 2000 mg/kg body weight/d dose level.

Lastly, treatment-related effects were observed during microscopical examination of the parental generation. Inflammation of the gastro-intestinal tract (increase in both incidence and severity of neutrophilic infiltrates, acute inflammation, goblet / epithelial cell hyperplasia, foci of brown pigment and/or oedema of the rectum, colon and cecum) was observed at the 1000 and 2000 mg/kg bw/day for both sexes and this finding was considered to be treatment-related.

During the observation of the offspring animals (F1), no test item-related effects were observed for clinical signs, mortality, viability, bodyweight and weight changes, gross pathology and sex ratio.

The NOAEL for reproductive/developmental toxicity cannot be determined, based to the absence of adverse toxic effects in all investigated reproductive/developmental parameters. Based on the highest dose tested, a dose of FemTA 2000 mg/kg body weight/day (equivalent to Fe(III) 80 mg/kg bw/d) could be considered to cause no adverse reproductive/developmental effects.

Based on the histopathological findings noted in the gastro-intestinal tract of male and female rats of the parental generation at the 1000 and 2000 mg/kg bw/day dose levels, the no observed adverse effect level (NOAEL) for general toxicity is considered to be 500 mg/kg bw/day for males and females. Furthermore, the NOAEL for developmental toxicity was determined to be 2000 mg/kg bw/day based on the absence of adverse toxic effects.

This reference had several minor reporting and experimental deficiencies:
First, the test substance is mixture composition of sodium tartrate, mesotartaric acid, chloride, iron, sodium, sodium oxalate, and water. Therefore, it is unclear, if the test item-related effects observed in the study are caused by the iron in the mixture or maybe by another compound in the mixture. Furthermore, the low pH of 3.7 alone might cause the inflammation of the gastro-intestinal tract instead of the iron content of the mixture.

According to the guideline the duration of study, following acclimatisation, should be dependent on the female performance and should last approximately 54 days, [at least 14 days pre-mating, (up to) 14 days mating, 22 days gestation, 4 days lactation]. In the study acclimatisation and mating period of the rats were described but clear information of the duration were not provided. In addition, in this study the duration of the premating period was not clearly described. In the method section, it was stated to be 12 weeks. However, according to the provided body weight data it appeared to have been only 11 weeks. Further, in the method section it was described, that male rats were treated for 90/91 days, but the reported body weight data indicate a treatment period of approximately 98 days. A prolongation of the 14 days premating period is even beneficial to observe cumulative adverse effects of low magnitude influencing the female fertility and the entire spermatogenesis of male animals.

The dosage volume was not equal among the treatment groups. In the study, dosing was achieved by the adapting the administrated volume of the test substance. Due to the slight acidic pH (3.7) the irritating aspects of the test substance was in the focus of the study. In the case of irritating substances the guideline foresees no dilution of the test substance.

Pre-treatment data was not provided or mentioned in the study. According to the provided body weight gain data, the parental (P) rats have been investigated before the treatment.

Furthermore, the authors stated that besides the clinical signs reported, they observed other clinical signs, which were even distributed across treated and control group and were considered to be normal for the age and strain of rat. The type of clinical signs were not further clarified by the authors. Furthermore, it is not clearly stated, if detailed clinical examinations were carried out before treatment was started, as foreseen by the guideline.

Reflecting the more pronounced adverse effects in male rats described above it can be concluded that male rats could be more susceptible to the treatment. The different response of the female mice could be due to the different investigation time points, pregnancy, weaning and lactation. Females were not in a similar state as the males during blood sampling, following pathology and histopathological investigations. Females were sampled at necropsy during the lactation phase. This could explain the different susceptibility of male and female rates in response of the FemTA treatment.

In the offspring (F1) generation the suckling behaviour was not mentioned to be investigated. In most cases an alteration of the suckling behaviour should have had an impact of the body weight gain of the F1 animals. However, the body weight gain of the F1 treatment groups were normal compared to the F1 control group, indicating a normal suckling behaviour.

Lastly, historical control data or individual data were not provided. Since the historical control data was not provided, it is not possible to determine, if the findings were within or outside the range of normal biological variation of the rat strain. In addition, individual data would be helpful in order to determine, if the results contain outliners, which might influence the outcome of the results.