Diiron trioxide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The evidence for the lack of toxicity to reproduction of the iron oxides category is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

other justification

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: as reported in source record

Justification for type of information:

see attachment "Endpoint-specific read-across justification for the iron oxide category" in section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

> 201 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

iron

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 201 mg/kg bw/day

Based on:

element

Remarks:

iron

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Conclusions:

With regard to the reproduction/development of the parent animals, a NOAEL for reproductive toxicity of 201 mg Fe/kg bw/day was concluded for the male and female rats due to the absence of any relevant toxicological effects. Also, a No Observed Adverse Effect Level (NOAEL) of 201 mg Fe/kg bw/day was concluded for the offspring (F1 generation) based on the absence of any relevant toxicological effect.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In this dossier, the toxicity to reproduction is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all substances of the iron oxides category. Source information is obtained from studies with soluble iron substances, which constitutes an intrinsic conservatism. The assessment of the effects in humans of iron oxides is related to the assumption that iron oxides are of very low solubility is artificial bodyfluids and of very low systemic bioavailability. Further information on the read-across approach are given in the report attached to IUCLID section 13.2.

 

Toxicity to reproduction – animal data

Kim et al 2004 and Bae et al. 2005 report a combined repeated dose toxicity study with the reproduction/developmental toxicity screening, which was conducted in accordance with OECD 422 and under GLP. Groups of 15 male and 15 female Sprague Dawley rats were administered via gavage iron dichloride at dose levels of 125, 250 and 500 mg/kg bw/day (equivalent to 55, 110 and 220 mg Fe/kg bw/day). The male and female rats were treated daily for a duration of 42 days and 42 to 54 days, respectively. The treatment period included a two-week pre-mating period as well as mating period, gestation period and lasted up to lactation day 4. A control group without treatment was run concurrently. In addition, recovery groups of five male and five female rats were employed for the control group and the high dose group.

During the observations of the parental male and female rats, no test item-related effects were observed for food consumption, haematology, clinical biochemistry, urinalysis, neurobehaviour, reproductive function (testis and epididymis weight) and reproductive performance (pre-implantation lost, post-implantation loss and mating data). However, clinicals signs such as blackish stool and salivation were observed for both sexes at the 125, 250 and 500 mg/kg bw/day dose levels. Furthermore, in the early stages of administration of 500 mg/kg bw/day, cases of decrease in locomotion activity were found in both sexes, but these were recovered to normal states. The female rats were more sensitively affected than the male rats in locomotion activity decrease, paleness, emaciation and soiled perineal region. However, these symptoms were reversible within the test period. In addition, three female rats of the 500 mg/kg bw/day dose group were found dead on the days 38, 46 and 51. The cause of death was gastrointestinal damages by the test substance. At the 250 and 500 mg/kg bw/day dose levels, the rate of body weight gain of the parental male rats was significantly decreased (- 4.2 % to - 8.2 % and - 7.5 % to -14 %, respectively) compared to the control group and water consumption was increased for male and female parental animals at the 500 mg/kg bw/day dose level compared to the control group.

The investigation of the organ weights of the parental generation revealed that both absolute and relative weights of liver were statistically significantly increased in 250 and 500 mg/kg bw/day male groups and in 500 mg/kg bw/day female group. Also, for male rats, absolute adrenal glands weights were statistically significantly increased in 500 mg/kg bw/day group, and relative adrenal glands weights were increased in 250 and in 500 mg/kg bw/day group. Because of hemosiderin deposit in hepatocyte and hyperplasia of zona fasciculate in adrenal cortex, the increased weights of liver and adrenal glands were influenced by the test substance. In 125 mg/kg bw/day male group, liver weight did not differ from the control group, but adrenal glands weights (left) were decreased as compared to the control group. Furthermore, the following necropsy findings were caused by the test substance: severe diffuse haemorrhagic glandular stomach and severe distension of stomach in dead animals, and diffuse black coloured liver and haemorrhage with diffuse black pigmentation in scheduled necropsy of 500 mg/kg bw/day male group. For females, a case of mass of mesenteric lymph node was observed in 500 mg/kg bw/day group. Lastly, the histopathological examination showed that for groups of both sexes, hemosiderin deposit of hepatocyte and glandular, hyperplasia of zona fasciculate in adrenal cortex, hyperkeratosis of forestomach, hemosiderin deposit of glandular stomach, neutrophil infiltration of submucosa were observed at the 500 mg/kg bw/day dose level. These conditions were induced by the test substance and were weaker in females. There were no specific findings in the recovery groups.

The investigation of the offspring revealed that survival rates were within the normal range and, therefore, no test item-related effects on survival rate was observed. In addition, no test item-related effects were observed on body weight, gross pathological findings, litter size, birth rate and sex ratio.

Based on the test item-related effects on body weight gain, water consumption, organ weights (liver and adrenal gland), macroscopical findings and microscopical findings, the no observed adverse effect level (NOAEL) for general toxicity is 125 mg/kg bw/day for males. The NOAEL for general toxicity is 250 mg/kg bw/day for females based on mortality, water consumption, organ weight (liver) and microscopical findings. The NOAELs for reproductive toxicity and developmental toxicity cannot be determined due to the absence of adverse toxic effects up to the highest dose of 500 mg/kg bw/day.

The study results were only available in a brief publication by Bae et al and as extended study summary (including raw data) in the OECD SIAR for Iron. However, the reporting detail is insufficient so that the study was rated with reliability 4. Further details are reported in the IUCLID study record.

 

In a combined repeated dose toxicity study with the reproduction developmental toxicity screening test (published study report by the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Japan, 2002), groups of 12 male and 12 female Sprague-Dawley rats were administered iron sulfate heptahydrate via gavage at dose levels of 30, 100, 300 and 1000 mg/kg bw/day (equivalent to 6, 20, 60 and 201 mg Fe/kg bw/day). Males and females were treated with the substance for a duration of 49 days (14 days before mating and 35 days after mating) and 42 - 47 days (14 days before mating, throughout the mating period and the gestation period until lactation day 5), respectively. A vehicle control group was run concurrently. The study was conducted in accordance with OECD 422 and under GLP.

After oral administration of 30, 100, 300 and 1000 mg/kg/day of the test item no effects were observed on food consumption and gross pathology. General observation revealed salivation in males and females in the ≥300 mg/kg groups. This was transient and only observed immediately after administration, and there were no neurological symptoms such as convulsion or morphological changes to the salivary glands, and so the salivation was attributed to irritation by the test substance, and was not deemed to be a symptom of toxicity. After the administration 1000 mg/kg/day of the test item, one male and one female died. These animals had exhibited salivation on observation of general condition. Necropsy of the dead animals revealed adrenal hypertrophy in the male and pituitary tumour, atrophy of the thymus, dark red discolouration of the lungs and adrenal hypertrophy in the female. Histological examination revealed mineral deposition in the heart, congestion of the lungs and yellow-brown pigment deposition in the periportal hepatocytes in the male and congestion and oedema in the lungs and mineral deposition in the liver in the female.

In addition to the findings described above for the 1000 mg/kg/day dose group, body weights in the 1000 mg/kg group were somewhat low throughout the administration period in the males, and tended to be low in the late gestation period in the females. Furthermore, temporarily low food consumption was observed in males and females in this group. Urine tests revealed high urine volume and low specific gravity in males of the1000 mg/kg/day group, but no changes attributable to test item administration were observed in the females. Haematology tests revealed low RBC and APTT values, and high MCV, MCH and reticulocyte levels in males, but no changes attributable to administration were observed in the females. Blood biochemistry test revealed low total protein, albumin and Ca levels, and high ALT, γ-GTP and A/G levels in males and high γ-GTP and organic phosphorus levels in females. The necropsies revealed dark red spots and ulceration of the glandular stomach mucosa in males in the 1000 mg/kg group, but no changes caused by administration were observed in the females. Further, organ weight measurements revealed high absolute and relative adrenal weights and high relative liver weights in males in the 1000 mg/kg group, and high absolute and relative liver weights in females in the 1000 mg/kg group. Lastly, the histological investigation of the 1000 mg/kg/day group revealed that the thymus findings were atrophy of the thymus in two males. The stomach findings were ulceration of the glandular stomach in one male, erosion of the glandular stomach in one male, inflammatory cell infiltration of the glandular stomach submucosa in two males, haemorrhage of the glandular stomach submucosa in one male, and vacuolisation of the forestomach epithelium in one male. The liver findings were yellow-brown pigment deposition in periportal hepatocytes in all six males, and yellow-brown pigment deposition in periportal Kupffer cells in three males and yellow-brown pigment deposition in periportal hepatocytes in all six females. The spleen findings were extramedullary haematopoiesis in four males, and yellow-brown pigment deposition in the red pulp in all six males and yellow-brown pigment deposition in the red pulp in all six females. These findings were observed at greater severity in the high dose group than in the control group. The kidney findings were basophilic changes in the tubular epithelium in four males. The bone marrow findings were increased haematopoiesis in the femur in one male. High organic phosphorus levels were seen in females of the 300 mg/kg group. Furthermore, the histopathological investigation revealed increased extramedullary haematopoiesis in the spleen in five males.

With regard to the reproduction/development of the parent animals, no histopathological changes were observed in the testes, epididymis, seminal vesicles, prostate, ovaries, uterus, vagina or mammary glands at any dose level. Moreover, no changes due to administration were observed in the number of oestrus, copulation index, number of days required for copulation, conception index, gestation index, nursing, lactation, number of corpora lutea, number of implantations, implantation index or gestation period. In the pups, no changes due to administration were observed in the total number of pups born, number of stillbirths, number of pups on lactation day 0, sex ratio on lactation day 0, delivery index, birth index, or live birth index. No changes due to administration were observed in the general condition of the pups. No changes due to administration were observed in the number of live pups on lactation day 4, sex ratio of the live pups on lactation day 4, or viability on lactation day 4. External observation revealed no changes due to administration. No changes due to administration were observed in the body weights. The necropsies of the pups revealed no changes due to administration.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of 300 mg/kg/day (equivalent to 60 mg Fe/kg bw/day) is derived for both sexes of the parental generation based on the increased relative liver weight and increased gamma glutamylpeptidase in males and females at the 1000 mg/kg/day dose level. With regard to the reproduction/development of the parent animals, a NOAEL for reproductive toxicity of 1000 mg/kg/day (equivalent to 201 mg Fe/kg bw/day) was concluded for the male and female rats due to the absence of any relevant toxicological effects. Also, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day (equivalent to 201 mg Fe/kg bw/day) was concluded for the offspring (F1 generation) based on the absence of any relevant toxicological effect. The study is considered as reliable with restriction (RL2), details on the shortcomings are reported in the IUCLID study record.

 

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/day (equivalent to 20, 40, or 80 mg of iron/kg body weight/day). 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. The study was conducted in accordance with OECD 422/408 and under GLP.

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.

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.

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, due to an absence of adverse toxic effects in all investigated reproductive/developmental parameters. Thus, 2000 mg/kg body weight/day (equivalent to Fe(III) 80 mg/kg bw/d) is the no adverse effect level for 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 500 mg/kg bw/day for males and females. The study is considered as reliable with restriction (RL2), details on the shortcomings are reported in the IUCLID study record.

 

Toxicity to reproduction – human data

The meta-analysis by Pena-Rosas et al. 2015a included 61 randomised and quasi-randomised controlled trials carried out since 1936 in 27 countries, mostly conducted during the last 20 years. The review included 27 trials from 15 countries, but only 21 trials (with 5490 women) contributed data to the review. All studies compared daily versus intermittent iron supplementation. The methodological quality of included studies was mixed and most had high levels of attrition. Of the 21 trials contributing data, three studies provided intermittent iron alone, 14 intermittent iron + folic acid and four intermittent iron plus multiple vitamins and minerals in comparison with the same composition of supplements provided in a daily regimen.

The objective of this meta-analysis was to assess the benefits and harms of intermittent supplementation with iron alone or in combination with folic acid or other vitamins and minerals to pregnant women on neonatal and pregnancy outcomes.

Preventive iron supplementation reduced maternal anaemia at term by 70% (risk ratio (RR) 0.30; 95% confidence interval (CI) 0.19 to 0.46, 14 trials, 2199 women, low quality evidence), iron-deficiency anaemia at term (RR 0.33; 95% CI 0.16 to 0.69, six trials, 1088 women), and iron deficiency at term by 57% (RR 0.43; 95% CI 0.27 to 0.66, seven trials, 1256 women, low quality evidence). There were no clear differences between groups for severe anaemia in the second or third trimester, or maternal infection during pregnancy (RR 0.22; 95% CI 0.01 to 3.20, nine trials, 2125 women, very low quality evidence; and, RR 1.21; 95% CI 0.33 to 4.46; one trial, 727 women, low quality evidence, respectively), or maternal mortality (RR 0.33; 95% CI 0.01 to 8.19, two trials, 12,560 women, very low quality evidence), or reporting of side effects (RR 1.29; 95% CI 0.83 to 2.02, 11 trials, 2423 women, very low quality evidence). Women receiving iron were on average more likely to have higher haemoglobin (Hb) concentrations at term and in the postpartum period but were at increased risk of Hb concentrations greater than 130 g/L during pregnancy, and at term.

Compared with controls, women taking iron supplements less frequently had low birthweight new-borns (8.4% versus 10.3%, average RR 0.84; 95% CI 0.69 to 1.03, 11 trials, 17,613 women, low quality evidence), and preterm babies (RR 0.93; 95% CI 0.84 to 1.03, 13 trials, 19,286 women, moderate quality evidence). They appeared to also deliver slightly heavier babies (mean difference (MD) 23.75; 95% CI -3.02 to 50.51, 15 trials, 18,590 women, moderate quality evidence). None of these results were statistically significant. There were no clear differences between groups for neonatal death (RR 0.91; 95% CI 0.71 to 1.18, four trials, 16,603 infants, low quality evidence), or congenital anomalies (RR 0.88, 95% CI 0.58 to 1.33, four trials, 14,636 infants, low quality evidence).

Findings of this meta-analysis suggest that intermittent regimens produced similar maternal and infant outcomes as daily supplementation but were associated with fewer side effects and reduced the risk of high levels of Hb in mid and late pregnancy, although the risk of mild anaemia near term was increased.

 

 

The meta-analysis by Pena-Rosas et al. 2015b included 61 randomised and quasi-randomised controlled trials carried out since 1936 in 27 countries, mostly conducted during the last 20 years. The review included 27 trials from 15 countries, but only 21 trials (with 5490 women) contributed data to the review. All studies compared daily versus intermittent iron supplementation. The methodological quality of included studies was mixed and most had high levels of attrition. Of the 21 trials contributing data, three studies provided intermittent iron alone, 14 intermittent iron + folic acid and four intermittent iron plus multiple vitamins and minerals in comparison with the same composition of supplements provided in a daily regimen.

The objective of this meta-analysis was to assess the effects of daily oral iron supplements for pregnant women, either alone or in conjunction with folic acid, or with other vitamins and minerals as a public health intervention in antenatal care.

Preventive iron supplementation reduced maternal anaemia at term by 70% (risk ratio (RR) 0.30; 95% confidence interval (CI) 0.19 to 0.46, 14 trials, 2199 women, low quality evidence), iron-deficiency anaemia at term (RR 0.33; 95% CI 0.16 to 0.69, six trials, 1088 women), and iron deficiency at term by 57% (RR 0.43; 95% CI 0.27 to 0.66, seven trials, 1256 women, low quality evidence). There were no clear differences between groups for severe anaemia in the second or third trimester, or maternal infection during pregnancy (RR 0.22; 95% CI 0.01 to 3.20, nine trials, 2125 women, very low quality evidence; and, RR 1.21; 95% CI 0.33 to 4.46; one trial, 727 women, low quality evidence, respectively), or maternal mortality (RR 0.33; 95% CI 0.01 to 8.19, two trials, 12,560 women, very low quality evidence), or reporting of side effects (RR 1.29; 95% CI 0.83 to 2.02, 11 trials, 2423 women, very low quality evidence). Women receiving iron were on average more likely to have higher haemoglobin (Hb) concentrations at term and in the postpartum period, but were at increased risk of Hb concentrations greater than 130 g/L during pregnancy, and at term.

Compared with controls, women taking iron supplements less frequently had low birthweight newborns (8.4% versus 10.3%, average RR 0.84; 95% CI 0.69 to 1.03, 11 trials, 17,613 women, low quality evidence), and preterm babies (RR 0.93; 95% CI 0.84 to 1.03, 13 trials, 19,286 women, moderate quality evidence). They appeared to also deliver slightly heavier babies (mean difference (MD) 23.75; 95% CI -3.02 to 50.51, 15 trials, 18,590 women, moderate quality evidence). None of these results were statistically significant. There were no clear differences between groups for neonatal death (RR 0.91; 95% CI 0.71 to 1.18, four trials, 16,603 infants, low quality evidence), or congenital anomalies (RR 0.88, 95% CI 0.58 to 1.33, four trials, 14,636 infants, low quality evidence).

Findings of this meta-analysis suggest that daily iron supplementation reduces the risk of maternal anaemia and iron deficiency in pregnancy but the positive effect on other maternal and infant outcomes is less clear.

 

Toxicity to reproduction – Conclusion

In the case of iron oxide category substances, the weight of evidence information demonstrates that these substances are extremely poorly soluble in water, show very low in vitro bioaccessibility over a whole range of surrogate biological media and are poorly bioavailable in vivo in rats. Detailed toxicokinetic investigations did not reveal any significant absorption and excretion. Iron oxide category substances showed very little gastrointestinal absorption. The low order of toxicity to reproduction is manifested (i) in an absence of concern in screening studies in the rat for the soluble iron substances and (ii) an absence of any adverse effects on organs of reproduction in any repeated dose toxicity studies with the iron oxide category substances reported to date. Two large meta-analysis studies did not show any adverse effects in pregnancy outcome upon iron supplementation.

The evidence for the lack of toxicity to reproduction of the iron oxides category is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.

Overall, the conduct of toxicity for reproduction studies with a source substance of the iron oxide category cannot be expected to contribute any relevant information to the assessment of (otherwise negative) information on reproduction toxicity.

Effects on developmental toxicity

Description of key information

The evidence for the lack of developmental toxicity of the iron oxides category is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Species:

rat

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Species:

rabbit

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

other: human studies as described in IUCLID section 7.10

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In this dossier, the developmental toxicity is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all substances of the iron oxides category. Source information is obtained from studies with soluble iron substances, which constitutes an intrinsic conservatism. The assessment of the effects in humans of iron oxides is related to the assumption that iron oxides are of very low solubility is artificial bodyfluids and of very low systemic bioavailability. Further information on the read-across approach are given in the report attached to IUCLID section 13.2.

 

Developmental toxicity – animal data

No higher-tier developmental toxicity study in animals is available for any member of the iron oxide category. However, there is sufficient weight of evidence from three high-quality guideline and GLP-compliant combined repeated dose toxicity study with the reproduction developmental toxicity screening test available. In all three studies highly soluble iron substances were administered to rats up to the highest dose of 2000 mg/kg bw/day. No signs of developmental toxicity was observed in any of the animals.

 

Developmental toxicity – human data

The meta-analysis by Haider et al. 2013 examined the exposure-response relation of dose of iron, duration of use, and haemoglobin concentration on birth weight and risk of low birth weight and preterm birth. Various comparisons were made evaluating the overall effect of iron, iron only, and iron with folic acid. In total 48 randomised trials (17,793 women) and 44 cohort studies (1,851,682 women) were included.

The randomised trials in this meta-analysis included studies in pregnant women of daily oral iron or iron and folic acid use compared with placebo, no iron, or no iron and folic acid. Trials of both supplementation and fortification were included. The analysis excluded trials of multiple vitamins and minerals, except those that examined the additional effect of iron or iron with folic acid in which all treatment groups received similar vitamins and minerals (except for iron or iron and folic acid). Trials examining maternal haematological, morbidity, and birth outcomes were included. Birth outcomes included mean duration of gestation (weeks), preterm birth (defined as birth of a neonate <37 weeks of gestation), mean birth weight (g), low birth weight (defined as birth weight <2500 g), mean birth length (cm), small for gestational age birth (defined as birth weight below the 10th centile of the gestational age and sex), stillbirth (defined as death of a fetus after 28 weeks of gestation), perinatal mortality (defined as deaths including stillbirths and neonatal deaths before 7 days of life), and neonatal mortality (defined as death of a neonate in the first month of life).

The observational studies in this meta-analysis included prospective cohort studies that allowed examination of the association of baseline anaemia with the above specified birth outcomes. Cross sectional and case-control studies were excluded, as these do not allow assessment of the temporal association between exposure and an outcome.

The meta-analysis showed that iron use increased maternal mean haemoglobin concentration by 4.59 (95% confidence interval 3.72 to 5.46) g/L compared with controls and significantly reduced the risk of anaemia (relative risk 0.50, 0.42 to 0.59), iron deficiency (0.59, 0.46 to 0.79), iron deficiency anaemia (0.40, 0.26 to 0.60), and low birth weight (0.81, 0.71 to 0.93). The effect of iron on preterm birth was not significant (relative risk 0.84, 0.68 to 1.03). Analysis of cohort studies showed a significantly higher risk of low birth weight (adjusted odds ratio 1.29, 1.09 to 1.53) and preterm birth (1.21, 1.13 to 1.30) with anaemia in the first or second trimester. Exposure-response analysis indicated that for every 10 mg increase in iron dose/day, up to 66 mg/day, the relative risk of maternal anaemia was 0.88 (0.84 to 0.92) (P for linear trend<0.001). Birth weight increased by 15.1 (6.0 to 24.2) g (P for linear trend=0.005) and risk of low birth weight decreased by 3% (relative risk 0.97, 0.95 to 0.98) for every 10 mg increase in dose/day (P for linear trend<0.001). Duration of use was not significantly associated with the outcomes after adjustment for dose. Furthermore, for each 1 g/L increase in mean haemoglobin, birth weight increased by 14.0 (6.8 to 21.8) g (P for linear trend=0.002); however, mean haemoglobin was not associated with the risk of low birth weight and preterm birth. No evidence of a significant effect on duration of gestation, small for gestational age births, and birth length was noted.

 

In a longitudinal study series on iron-deficiency anaemia and behaviour/cognition (Walter et al. 1998; Lozoff et al. 2012; Gahagan et al. 2019; East et al. 2020), infants were recruited from community clinics in low- to middle-income neighbourhoods in Santiago, Chile. Six-month-old infants were randomized to iron-fortified (12.7 mg/L) or low-iron (2.3 mg/L) formula for six months. The formula contained elemental iron as ferrous sulfate. The iron status (haemoglobin, mean cell volume, erythrocyte protoporphyrin and serum ferritin) of the infants was determined at 12 months of age. In addition, the iron status of the low-iron group was determined at 18 months of age. A follow-up study was conducted when the children were 10 years of age. During this follow-up study IQ, spatial memory, arithmetic achievement, visual-motor integration, visual perception, and motor functioning were measured. Furthermore. a covaried regression was used to compare iron-fortified and low-iron groups and considered haemoglobin level before randomization and sensitivity analyses to identify 6-month haemoglobin levels at which groups diverged in outcome. Another follow-up study was conducted at 16 years of age. At 16 years of age, cognitive ability, visual perceptual ability, visual memory and achievement in math, vocabulary, and comprehension were assessed, using standardized measures. Mean differences in developmental test scores were compared according to randomization group. Lastly, at age of 21 years, the young adults who took part in the trial during infancy were again investigated. The adults were assessed for neurocognition, emotion regulation, educational level, and attainment of adult developmental milestones.

In this study, low-iron formula was not significantly inferior to iron-fortified formula in the prevention of iron-deficiency anaemia. At 12 months of age, 3.8 % of the low-iron group had iron-deficiency anaemia compared to 2.8 % of the high-iron group (p = 0.35). However, a greater proportion of infants in the low-iron group was iron deficient at 12 months of age by the criterion of 2 of 3 measures of iron status in the deficient range, but mean differences in iron measures were minor from a clinical perspective. Infants in the low-iron group did not progress to iron-deficiency anaemia. All indicators of iron status improved in the low-iron group by 18 months of age. Infants in the high-iron group had higher 12-month haemoglobin and ferritin levels and mean cell volumes, and lower levels of erythrocyte protoporphyrin than those in the low-iron group (p < 0.005). This indicated that the amount of iron in the formula did affect overall iron status.

Children who received 12.7 mg/L of iron-fortified formula as infants had lower cognitive and visual-motor scores at 10 years than those receiving low-iron formula. However, the differences were only observed among children with the very highest or lowest haemoglobin levels on entry into the trial at 6 months. Children with high haemoglobin levels had lower 10-year test scores if they received iron-fortified formula, whereas those with low haemoglobin levels had higher scores. In conclusion, this study indicates poorer long-term developmental outcome in infants with high haemoglobin concentrations who received formula fortified with iron. Most infants showed no developmental effects of iron-fortified formula, and those with low haemoglobin levels in infancy had higher 10-year test scores if they received iron-fortified formula.

Adolescents who were randomized to iron-fortified formula (12 mg/L) between 6 and 12 months had lower cognitive scores, at 16 years, compared to those who had received low-iron formula (2.3 mg/L). The low-iron group performed better than the iron-fortified group on eight out of nine measures, with statistically significant differences in verbal comprehension, arithmetic achievement, and spatial memory. Moreover, there was no impact on these findings when infants who became iron deficiency anaemia (IDA) or were treated with oral iron therapy were excluded from the analyses. Furthermore, an interaction between 6-month haemoglobin status and iron supplementation at 16 years was found, but only for visual motor integration (VMI). Participants who had low 6-month haemoglobin had higher scores for VMI if they had been randomized to iron-fortified formula and those who had high 6-month haemoglobin had higher scores for VMI if they had been randomized to low-iron formula.

This study found that lower age 10 cognitive abilities stemming from consuming iron-fortified formula in infancy were associated with poorer neurocognitive functioning, emotional awareness, and educational attainment in young adulthood. Additionally, consumption of iron-fortified formula was marginally directly related to poorer verbal memory and slower mental processing in young adulthood, and modest dosage effects of iron-fortified formula were found for lower educational attainment and slower processing speed. In the current study, the children at age 10 tested broadly in the normal intelligence range, with those receiving iron-fortified formula scoring in the slightly lower normal range than those receiving low-iron supplementation. Individuals who received iron-fortified formula in infancy and had relatively low IQ and low spatial memory at age 10 had the poorest neurocognitive performance as young adults. Lastly, results of the regression analyses also showed that participants who received iron-fortified formula and had average above average age 10 IQ or spatial memory scored equally as well on neurocognitive tests as those or who received low-iron formula in infancy.

The longitudinal study, as described above, has experimental and reporting deficiencies. First of all, during the treatment period it was not recorded, if the infants received solid food besides the diet formula and breast feeding and what kind of solid food they were given to eat. Therefore, it cannot be ruled out that the infants also received iron from other food source and the overall amount of iron consumed by the infants might have been higher or lower as assumed. In addition, as stated by East et al. (2020), differences in performance between the iron-fortified and low-iron groups could be the results of an interaction between the level of iron and another formula component. Furthermore, as pointed out by Gahagan et al. (2019), the iron absorption was not measured and the exact amount of iron metabolised is unknown.

In this longitudinal study, haemoglobin levels were measured in infants in order to determine the iron status, however, there also are other measures of iron status that could be investigated to obtain more precise measurement of iron. For example, serum iron could be determined, since not all iron will be bound by haemoglobin. Furthermore, as pointed out by Gahagan et al. (2019), venous measurements of haemoglobin would have been more accurate as capillary haemoglobin measurement. Since infants were only investigated by venous measurements when the capillary measurements indicated a problem with the haemoglobin level, it might be that infants were included in the study which would have been excluded otherwise, if venous measurements would have been used.

Confounding factors such as smoking by the mothers or other household members were not controlled for in the study. Also, the possibility of alcohol consumption by the mothers during pregnancy was not determined. Such factors might also affected the developmental outcome. In addition, the authors did not determine the iron status prenatally or neonatally, as mentioned by Gahagan et al. (2019). Therefore, it could be that the iron status at that time might also play a role in the outcome of the study. Although adjusted estimates from randomised trails and cohort studies were used, these data were of observational nature. These associations still could have been biased owing to residual confounding, in either direction, depending on the nature of the residual confounding. Associations with several outcomes could not be evaluated owing to the paucity of data (stillbirths, neonatal and perinatal mortality in iron use meta-analyses; birth length, neonatal mortality in the cohort studies analysis). A small number of trials had evaluated the effect of iron fortification in pregnant women, so a separate meta-analysis for fortification trials could not be done. Significant heterogeneity existed for several outcomes that could not be explained substantially by pre-specified subgroups. This limits the understanding of the association in various settings and restricts the generalisability of the findings. For the exposure-response analysis of cohort studies, mean haemoglobin concentrations for most studies were missing. An assumption was made, that countries within the same category (low-, middle-, or high-income) would have similar mean haemoglobin concentrations. These mean values were used for analysis, which may have introduced bias towards the null due to random measurement error.

 

Developmental toxicity – Conclusion

In the case of iron oxide category substances, the weight of evidence information demonstrates that these substances are extremely poorly soluble in water, show very low in vitro bioaccessibility over a whole range of surrogate biological media and are poorly bioavailable in vivo in rats. Detailed toxicokinetic investigations did not reveal any significant absorption and excretion. Iron oxide category substances showed very little gastrointestinal absorption. The low order of developmental toxicity is manifested (i) in an absence of concern in screening studies in the rat for the soluble iron substances and (ii) an absence of any adverse effects on organs of reproduction in any repeated dose toxicity studies with the iron oxide category substances. A large meta-analysis study did not show any adverse effects in the development during pregnancy and post-partum upon iron supplementation.

The evidence for the lack of developmental toxicity of the iron oxides category is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.

Overall, the conduct of developmental toxicity studies with a source substance of the iron oxide category cannot be expected to contribute any relevant information to the assessment of (otherwise negative) information on developmental toxicity.