Iron(II) oxalate

Endpoint summary

Description of key information

A 90 days study (OECD 408) was conducted on wistar rat (male/female) to assess the toxicity of oxalic acid via oral route. This study is used in a read across approach to assess the toxicity of iron oxalate.

No treatment related effects were observed (e.g. functional observation battery, hematology, clinical biochemistry, adrenals, and thymus). The effects observed were due to species, strain, age, biological variation, incidental, congenital and/or physiology related.

No Observed Adverse Effect Level (NOAEL) –  1000 ppm. This value corresponds to 63 mg/kg bw/d, according to calculations

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

January 06, 2016 to March 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information in a detailed justification report is included as attachment to the same record.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For the determination of analogue in this read-across approach, the following points have been considered:
- Chemical speciation and valency (common valency of the cation: Fe2+ compared to Mg2+ or Sr2+).
- The water solubility, as it provides a first indication of the availability of the metal ion in the different compartments of interest. The most simplistic approach to hazard evaluation is to assume that the specific metal-containing compound to be evaluated shows the same hazards as the most water-soluble compounds.
- In fluids of organisms and in aqueous media, dissociation of ferrous oxalate di hydrate takes place immediately, resulting in formation of Ferrous cations (Fe2+) and oxalate anions. Thus, any ingestion or absorption of ferrous oxalate di hydrate by living organisms, in case of systemic consideration, will inevitably result of exposure to the dissociation products.
- Iron is an abundant mineral naturally present in the body. Physiologically, it. exists as an ion in the body as Fe2+ (ferrous ion). It is a necessary trace element used by all known living organisms (Williams 2012, NCBI 2019). Iron-containing enzymes, usually containing heme prosthetic groups, participate in catalysis of oxidation reactions in biology, and in transport of a number of soluble gases (Fraùsto da Silva 2001, NCBI 2019). Iron is an essential constituent of hemoglobin, cytochrome, and other components of respiratory enzyme systems. Its chief functions are in the transport of oxygen to tissue (hemoglobin) and in cellular oxidation mechanisms. Inorganic iron involved in redox reactions is also found in the iron-sulfur clusters of many enzymes, such as nitrogenase (involved in the synthesis of ammonia from nitrogen and hydrogen) and hydrogenase (NCBI 2019). A class of non-heme iron proteins is responsible for a wide range of functions such as ribonucleotide reductase (reduces ribose to deoxyribose; DNA biosynthesis) and purple acid phosphatase (hydrolysis of phosphate esters).
- Counter ions: the assumption that the oxalate ion is responsible for the common property or effect implies that the toxicity or ecotoxicity of the counter ion present in the compound will be largely irrelevant in producing the effects to be assessed.
- Likely common breakdown products via physical and/or biological processes for the targeted substance (ferrous oxalate di hydrate) and the analogues identified cannot present strong differences since the structures are very simple and very similar (formation of Fe2+ or oxalate ion).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical information is provided in the “source” endpoint. No impurity affecting the classification is reported for the source chemical.
Information on the impurities of the target chemical are detailed in the attached report.

3. ANALOGUE APPROACH JUSTIFICATION
The main hypothesis for the analogue approach are verified. They are presented in the detailed report attached. The experimental data performed on the substance (tests performed in this REACH registration) confirms the analogue approach performed (same results on analogues).

4. DATA MATRIX
A data matrix is presented in the detailed report attached.


References :
Fraùsto da Silva J.J.R., Williams R.J.P. 2001 The biological chemistry of the elements, 2nd edition. Oxford University Press, Oxford
NCBI, National Center for Biotechnology Information. PubChem Compound Database; CID=27284, https://pubchem.ncbi.nlm.nih.gov/compound/27284 (accessed Mar. 6, 2019).
Williams R.J.P 2012. Iron in evolution. FEBS Letters. Volume 586, Issue 5, 9 March 2012, Pages 479-484.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in any of the animals treated with test item at the doses of 250 (low dose), 500 (intermediate dose) and 1000 PPM (high dose) throughout the treatment period. No clinical signs were observed in any of the animals during recovery period

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in any of the animal treated with test item at dose levels of 250, 500 and 1000 PPM, throughout the experimental and recovery periods

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the 90 consecutive days of treatment period, the body weight (g) and body weight gain (%) in animals across all the dose groups was found to be non significant when compared with control animals. No significant difference were observed in high dose recovery group when compared with control recovery group.
The body weight of all the animals was within the normal range of variability commonly recorded for this species, strain and age

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During the 90 consecutive days of dosing period, the quantity of feed consumed by animals across different dose groups was found to be non significant and comparable with the control animals. There was significant increase in feed consumption in male recovery (G4R) animals at weeks 3, 4 and 8 and significant decrease at weeks 15 and 16 when compared with control recovery group (G1R). In female recovery (G4R) animals, there was significant decrease at week 16 when compared with control recovery group (G1R)

OXALIC ACID was administered to Wistar rats at the doses of 0, 250, 500 and 1000 PPM in the diet for 90 days. The calculated mean intake of test item at the doses of 0, 250, 500 and 1000 PPM were equivalent to 0, 16, 31 and 63 mg/kg body weight/day respectively. This was selected since in the dose range finding study at 10000 and 5000 PPM, lesions in the kidney were noted.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Opthalmological examiniation did not reveal any abnormalities during acclimatization and in the control and high dose treated animals, at the end of treatment period

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology parameters viz Erythrocyte count (RBC), Hemoglobin (Hb), Hematocrit (PCV), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC) and Differential leukocyte count (DC) did not show any toxicologically relevent findings in both the sexes. In males, there was significant decrease in platelet (thrombocyte) count (PLT) (G3) and white blood cell count (WBC) (G2 and G4) as compared with control group (G1). In females, there was significant decrease in white blood cell count (WBC) (G4) as compared with intermediate dose group (G3). Significant increase in neutrophils (G4) as compared with low dose group (G2). Significant decrease in lymphocytes (G4) as compared with low dose group (G2). Significant increase in basophils (G4) as compared with intermediate dose group (G3). Significant decrease in prothrombin time (PT) (G4) as compared with control (G1) and low dose group (G2) and there was significant increase in activated partial thromboplastin time (APTT) (G3) as compared with control group (G1) at the end of week 13. In recovery males, there was significant decrease in white blood cell count (WBC) (G4R) as compared with control recovery (G1R) at the end of week 17. The changes observed in the hematological parameters are marginal and could not be attributed to the test item administartion as these values were within biological variation

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters viz Glucose (GLU), Urea, BUN, Calcium, Cholesterol (CHOL), Triglycerides (TRIGL), Alanine aminotransferase (ALT), Bilirubin (BIL), Sodium (Na+), Total protein (TPO) and Globulin (GLB) did not show any toxicologically releavant findings in both the sexes in treatment and recovery groups. In males, there was significant decrease in chloride (Cl-) (G2) as compared with control group at the end of week 13. in females, there was significant increase in alkaline phosphatase (ALP) (G4) when compare with control (G1), low (G2) and intermediate (G3) dose groups and significant decrease in chloride (Cl-) (G2) as compared with control group at the end of week 13. In recovery males, there was significant decrease in aspartate aminotransferase (AST) and phosphorous (PHOS) (G4R) when compared with control recovery (G1R) group. Significant increase in albumin (ALB) and albumin/globulin ratio (A/G) (G4R) when compared with control recovery (G1R) group. In recovery females, significant increase in potassium (K+) and creatinine (CREA) (G4R) when compared with control recovery (G1R) group at th end of week 17. The changes observed in the clinical biochemistry parameters are marginal and could not be attributed to the test item administartion as these values were within biological variation

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urine analysis parameters (volume, specific gravity, colour, clarity, pH, Erythrocytes, urobillinogen, bilirubin, proteins and glucose and microscopic examination) did not reveal any test item related changes in both the sexes during treatment and recovery periods. In males, there was significant increase in crystals (G3) when compared with control group (G1) at the end of week 13. In recovery males, there was significant increase in ketone bodies, leukocytes, pus cells and crystals (G4R) when compared with control recovery group (G1R) at the end of week 17

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation battery parameters viz grip strength (Forelimbs and Hind limbs), Rotarod, Urine pools, Rearing, Fecal bolus, Hind limb foot splay and IR Actimeter did not show any toxicological significant changes in all the treated groups when compared with control group. In treatment males, there was significant decrease in forelimbs of grip strength (G3) when compared with control group (G1). In treatment females, there was significant increase in rearing (G3) when compared with control group (G1). In recovery males, there was significant increase in Rotarod, urine pools and IR Actimeter (G4R) when compared with control recovery group (G1R) at the end of week 13

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related statistical significance was observed in absolute and relative organ weights of male animals during treatment and recovery period. In treatment groups, significantly higher absolute and relative weight of adrenals was observed in male animals of intermediate dose (G3) group compared to control (G1) group. No significant difference was observed in absolute and relative weight of female animals. In recovery groups, significantly lower absolute and relative weight of thymus was observed in male animals of high dose recovery (G4R) group compared to control recovery (G1R) group. In female animals, significantly lower absolute and relative weight of adrenals was observed in high dose recovery (G4R) group compared to control recovery (G1R) group and significantly lower absolute weight of brain in high dose recovery (G4R) group compared to control recovery (G1R) group. Despite these differences, microscopically no test item-related change was observed in these organs evaluated in treatment group. Hence, the changes observed in organ weight were considered to be due to changes in body weight or as biological variation

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy performed at the end of the treatment and recovery period, no test item related findings were observed macroscopically in all treated male and female animals. No gross abnormality was observed in control group animals. Macroscopically, small sized testes, epididymides, prostate, seminal vesicles with coagulating glands were observed in one male animal of intermediate dose (G3) group. Distended uterus with watery content was observed in five female animals each of control (G1) and of intermediate dose (G3) groups, six female animals of low dose (G2) and seven female animals of high dose (G4) group. In recovery groups, distended uterus with watery content was observed in six female animals of control recovery (G1R) and two female animals of high dose recovery (G4R) group. In addition, hydronephrosis was observed in one female animal of control recovery (G1R) group. These findings were considered as incidental and to be gender, congenital and/or physiology related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination revealed no abnormality attributable to the test item OXALIC ACID in high dose (1000 PPM) group. Most of the tissues evaluated were within normal histological limits. The histopathological findings observed in various tissues during evaluation of test item group animals were comparable with control group and were considered incidental. These changes observed can usually be considered to be species, age, gender, physiological or mode of death related and are covered in background historical data of pathology

Details on results:

No Observed Adverse Effect Level (NOAEL) – 1000 PPM

Applying the formula:

RFC = AFC/7 + [bw start of week 1 + bw gain during week/2]

Is obtained an Average test item consumed during the 90 day study of = 63 mg/kg bw day

Since no adverse effects were observed at highest tested dose i.e., 1000 PPM, the same was selected as NOAEL. The 1000 PPM is equivalent to 63 mg/kg body weight (actual test item consumed by the animals).
Test item (Oxalic acid dehydrate) as is basis = 63 mg/kg

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 63 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: No Observed Adverse Effect Level (NOAEL) – 1000 ppm. This value corresponds to 63 mg/kg bw/d, according to calculations accordig the formula: RFC (Relative Feed Consumption) = AFC/7 + [bw start of week 1 + bw gain during week/2]

Critical effects observed:

no

 Toxic response/effects by sex and dose level

 

Toxic response/effects	Dose Level	Sex
		Male	Female
Mortality/Viability	250 PPM   500 PPM   1000 PPM	No mortality	No mortality
Clinical Signs		No clinical signs	No clinical signs
Ophthalmoscopy		No abnormalities	No abnormalities
Body Weight		No Significance observed	No Significance observed
Body Weight Changes		No Significance observed	No Significance observed
Feed Consumption		Significance observed, but not treatment related	Significance observed, but not treatment related
Hematology		Significance observed, but not treatment related	Significance observed, but not treatment related
Biochemistry		Significance observed, but not treatment related	Significance observed, but not treatment related
Urinalysis		Significance observed, but not treatment related	No Significance observed
FOB		Significance observed, but not treatment related	No Significance observed
Organ Weights		Significance observed, but not treatment related	Significance observed, but not treatment related
Histopathology		Incidental changes, but not treatment related	Incidental changes, but not treatment related

Conclusions:

Repeated Dose 90 Days Dietary Toxicity Study with OXALIC ACID in Wistar Rats
This study was conducted based on the OECD Guidelines for the Testing of Chemicals, Section 4, Health Effects, Number 408, September 21, 1998.
The purpose of this study was to assess the toxicity of OXALIC ACID when administered to Wistar rats through diet for a period of 90 days. The reversibility of treatment related changes was assessed after a treatment free 28 day recovery period
Dose levels for main study are selected based on Dose Range Finding observation and results. Dose levels for main study were 250 PPM (low dose), 500 PPM (intermediate dose) and 1000 PPM (high dose). The test item was mixed with diet and administered to wistar rats. Each group consisting of ten males and ten females were used for the main study.
The following observations were performed: Mortality/viability, clinical signs, Ophthalmoscopy, body weights, feed consumption, dose formulation analysis, functional observation battery, clinical pathology, macroscopic and microscopic examination, organ weights and histopathology of the preserved tissues.
Statistical analysis was performed using Stat plus software to compare the significant difference between treatment and control group of animals (t-test/ANOVA).
Results:
No mortality was observed in any of the animal treated with test item at dose levels of 250, 500 and 1000 PPM, throughout the experimental and recovery periods.
No clinical signs were observed in any of the animals treated with test item at the doses of 250 (low dose), 500 (intermediate dose) and 1000 PPM (high dose) throughout the treatment period. No clinical signs were observed in any of the animals during recovery period.
Opthalmological examiniation did not reveal any abnormalities during acclimatization and in the control and high dose treated animals, at the end of treatment period.
During the 90 consecutive days of dosing period, the quantity of feed consumed by animals across different dose groups was found to be non significant and comparable with the control animals. There was significant increase in feed consumption in male recovery (G4R) animals at weeks 3, 4 and 8 and significant decrease at weeks 15 and 16 when compared with control recovery group (G1R). In female recovery (G4R) animals, there was significant decrease at week 16 when compared with control recovery group (G1R).
During the 90 consecutive days of treatment period, the body weight (g) and body weight gain (%) in animals across all the dose groups was found to be non significant when compared with control animals. No significant difference were observed in high dose recovery group when compared with control recovery group. The body weight of all the animals was within the normal range of variability commonly recorded for this species, strain and age.
Functional observation battery parameters viz grip strength (Forelimbs and Hind limbs), Rotarod, Urine pools, Rearing, Fecal bolus, Hind limb foot splay and IR Actimeter did not show any treatment related significant changes in all the treated groups when compared with control group. In treatment males, there was significant decrease in forelimbs of grip strength (G3) when compared with control group (G1). In treatment females, there was significant increase in rearing (G3) when compared with control group (G1). In recovery males, there was significant increase in Rotarod, urine pools and IR Actimeter (G4R) when compared with control recovery group (G1R) at the end of week 13.
Hematology parameters viz Erythrocyte count (RBC), Hemoglobin (Hb), Hematocrit (PCV), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC) and Differential leukocyte count (DC) did not show any toxicologically relevent findings in both the sexes. In males, there was significant decrease in platelet (thrombocyte) count (PLT) (G3) and white blood cell count (WBC) (G2 and G4) as compared with control group (G1). In females, there was significant decrease in white blood cell count (WBC) (G4) as compared with intermediate dose group (G3). Significant increase in neutrophils (G4) as compared with low dose group (G2). Significant decrease in lymphocytes (G4) as compared with low dose group (G2). Significant increase in basophils (G4) as compared with intermediate dose group (G3). Significant decrease in prothrombin time (PT) (G4) as compared with control (G1) and low dose group (G2) and there was significant increase in activated partial thromboplastin time (APTT) (G3) as compared with control group (G1) at the end of week 13. In recovery males, there was significant decrease in white blood cell count (WBC) (G4R) as compared with control recovery (G1R) at the end of week 17. The changes observed in the hematological parameters are marginal and could not be attributed to the test item administartion as these values were within biological variation.
Clinical biochemistry parameters viz Glucose (GLU), Urea, Cholesterol (CHOL), Triglycerides (TRIGL), Alanine aminotransferase (ALT), Bilirubin (BIL), Sodium (Na+), Total protein (TPO) and Globulin (GLB) did not show any toxicologically releavant findings in both the sexes in treatment and recovery groups. In males, there was significant decrease in chloride (Cl-) (G2) as compared with control group at the end of week 13. in females, there was significant increase in alkaline phosphatase (ALP) (G4) when compare with control (G1), low (G2) and intermediate (G3) dose groups and significant decrease in chloride (Cl-) (G2) as compared with control group at the end of week 13. In recovery males, there was significant decrease in aspartate aminotransferase (AST) and phosphorous (PHOS) (G4R) when compared with control recovery (G1R) group. Significant increase in albumin (ALB) and albumin/globulin ratio (A/G) (G4R) when compared with control recovery (G1R) group. In recovery females, significant increase in potassium (K+) and creatinine (CREA) (G4R) when compared with control recovery (G1R) group at th end of week 17. The changes observed in the clinical biochemistry parameters are marginal and could not be attributed to the test item administartion as these values were within biological variation.
Urine analysis parameters (volume, specific gravity, colour, clarity, pH, Erythrocytes, urobillinogen, bilirubin, proteins and glucose and microscopic examination) did not reveal any test item related changes in both the sexes during treatment and recovery periods. In males, there was significant increase in crystals (G3) when compared with control group (G1) at the end of week 13. In recovery males, there was significant increase in ketone bodies, leukocytes, pus cells and crystals (G4R) when compared with control recovery group (G1R) at the end of week 17.
No test item related statistical significance was observed in absolute and relative organ weights of male animals during treatment and recovery period. In treatment groups, significantly higher absolute and relative weight of adrenals was observed in male animals of intermediate dose (G3) group compared to control (G1) group. No significant difference was observed in absolute and relative weight of female animals. In recovery groups, significantly lower absolute and relative weight of thymus was observed in male animals of high dose recovery (G4R) group compared to control recovery (G1R) group. In female animals, significantly lower absolute and relative weight of adrenals was observed in high dose recovery (G4R) group compared to control recovery (G1R) group and significantly lower absolute weight of brain in high dose recovery (G4R) group compared to control recovery (G1R) group. Despite these differences, microscopically no test item-related change was observed in these organs evaluated in treatment group. Hence, the changes observed in organ weight were considered to be due to changes in body weight or as biological variation.
Necropsy performed at the end of the treatment and recovery period, no test item related findings were observed macroscopically in all treated male and female animals. No gross abnormality was observed in control group animals. Macroscopically, small sized testes, epididymides, prostate, seminal vesicles with coagulating glands were observed in one male animal of intermediate dose (G3) group. Distended uterus with watery content was observed in five female animals each of control (G1) and of intermediate dose (G3) groups, six female animals of low dose (G2) and seven female animals of high dose (G4) group. In recovery groups, distended uterus with watery content was observed in six female animals of control recovery (G1R) and two female animals of high dose recovery (G4R) group. In addition, hydronephrosis was observed in one female animal of control recovery (G1R) group. These findings were considered as incidental and to be gender, congenital and/or physiology related.
Microscopic examination revealed no abnormality attributable to the test item OXALIC ACID in high dose (1000 PPM) group. Most of the tissues evaluated were within normal histological limits. The histopathological findings observed in various tissues during evaluation of test item group animals were comparable with control group and were considered incidental. These changes observed can usually be considered to be species, age, gender, physiological or mode of death related and are covered in background historical data of pathology.
The analytical method was validated and the linearity response was found to be linear (r2 = 1.000) in the range of 10 to 500.1 mg.
The recovered content was found homogeneously distributed in the feed for main study at concentrations 250 mg/L, 500 mg/L and 1000 mg/L for the test item respectively. (i.e. % coefficient of variation (RSD) ranged in between 0.00 to 6.11) and for Dose range finding study at concentrations 1000 mg/L, 5000 mg/L and 10000 mg/L for the test item respectively. (i.e. % coefficient of variation (RSD) ranged in between 0.52 to 3.00).
The recovered content obtained in all the dose groups was in agreement with the target concentration (i.e. ranged between 91.42 % to 104.48 % for main study and 93.95 % to 97.19 % for dose range finding study).


CONCLUSION
OXALIC ACID was mixed with diet and administered to wistar rats for a period of 90 days at doses of 250, 500 and 1000 PPM. No test item related changes in the body weights, feed consumption, haematology, clinical biochemistry, urine parameters and organ weights were noted. Macroscopic and microscopic examination revealed no abnormality attributable to the treatment at the highest dose (1000 PPM). Hence, under the conditions of the experiment and based on the findings of the present study entitled “Repeated Dose 90 Days Oral Toxicity Study with OXALIC ACID in Wistar Rats” the NOAEL (No Observed Adverse Effect Level) was found to be the highest dose level employed i.e., 1000 PPM.

No treatment related effects were observed (e.g. functional observation battery, hematology, clinical biochemistry, adrenals, and thymus). The effects observed were due to species, strain, age, biological variation, incidental, congenital and/or physiology related.

No Observed Adverse Effect Level (NOAEL) – 1000 ppm. This value corresponds to 63 mg/kg bw/d, according to calculations accordig the formula: RFC (Relative Feed Consumption) = AFC/7 + [bw start of week 1 + bw gain during week/2]

1000 ppm corresponds to 50 mg/kg if you apply 0.05 factor. However this is applicable for more than 13 weeks study not for the below or up to 13 week study.
63 mg/kg of test item calculate using the above formula based on the actual amount feed consumed and bodyweight gain of animals. Hence there will be difference while calculating with factor and actual values obtained in the study.


These results are considered relevant in a read across approach and are used in the assessment of Iron Oxalate properties.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

63 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Justification for classification or non-classification

Since no adverse effects were observed at highest tested dose i.e., 1000 ppm, the same was selected as NOAEL, which is much higher than the cut-off value requiring a classification.