Iron(II) oxalate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A study was carried according to a protocol developed by the National Toxicology Program. This guideline is very similar to the two-generations study described by the OECD (OECD 416). The parameters checked are also in line with OECD 416. Not all raw data are present in the study report. The study has been perfomed in accordance with GLP. No reproductive effect related to the tet item was observed. These results are considered relevant and are used in a read across approach.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive 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:

Remarks:

The study was carried according to a protocol developed by the National Toxicology Program. This guideline is very similar to the two-generations study described by the OECD (OECD 416). The parameters checked are also in line with OECD 416. Not all raw data are present in the study report. The study has been perfomed in accordance with GLP.

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

Vehicle:

water

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
2 females died during cohabition of task 2 (1 in the 0,05% and 1 in the 0,1% dose group)
A dose relate decrease in water consumption was noted at 0,1% and 0,2% dose levels. However this did not result in any significant clinical toxicity

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No significant effect

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
no significant effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant effects. No effect on the relative frequency of different estrous stages exept for percent estrus (11% vs 21% in the treatment group). Additional experiments are needed to confirm the adverse effects of oxalic acid on the estrus phase.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No significant effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
Prostate gland in animals exposed to 0,2% oxalic acid was smaller as evidenced by significantly reduced absolute and adjusted weights at necropsy

GROSS PATHOLOGY (PARENTAL ANIMALS)
No significant effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
no significant effects

OTHER FINDINGS (PARENTAL ANIMALS)
Significant increase in the percentage of abnormal sperm

Key result

Dose descriptor:

NOAEL

Effect level:

<= 0.1 other: %

Sex:

male/female

Basis for effect level:

other: Fertility

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
Task 2: At the highest dose level tested a small but significant drop in the number of litters (4,7 vs 4,92) was noted.
Task 4: The total number of live pups and the number of live female pups delivered by second generation breeding pairs were significantly less than the corresponding control values

CLINICAL SIGNS (OFFSPRING)
No significant effects

BODY WEIGHT (OFFSPRING)
No significant effects

SEXUAL MATURATION (OFFSPRING)
No significant effects

ORGAN WEIGHTS (OFFSPRING)
The kidney weight of treated animals was significantly higher than the control group. When the organ weights were adjusted for body weight at necropsy, the observed differences with differences with respect to kidney weight were no longer significant.

GROSS PATHOLOGY (OFFSPRING)
No data

HISTOPATHOLOGY (OFFSPRING)
No data

OTHER FINDINGS (OFFSPRING)
Incidence of abnormal sperm was significantly higher than the control value.
Blood-serum calcium levels in treated animals and controls were essentially the same.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

<= 1 000 ppm

Sex:

male/female

Basis for effect level:

other: Fertility

Key result

Reproductive effects observed:

no

Table 3. Male Body and Organ Weights at Necropsy (Task 2) Oxalic Acid
	Treatment Group
Variable (a	Control	0,20%
Body (g)	40.270± 1.797(10) (b,c	44.840± 2.098 ( 10)
Liver. (g)	1.837 ± 0.082 ( 10)	2.015 ± 0.087 (10)
Kidneys (g) d	0.700± 0.025 ( 10)	0.729± 0.033 (°10)
Seminal Vesicles (g)	0.663± 0.047 ( 10)	0.688± 0.042 ( 10)
R. Testis (g)	0.134 ± 0.007 (10)	0.14'1 ± 0.012 (10)
R. Cauda (mg)	16.980± 0.778 (10)	17.750± 0.725(10)
R. Epididymis (mg)	51.250± 1.912(10)	50.770± 1.778 (10)
Prostate Gland (mg)	23.670 ± 1 .627 (10)	19.356 ± 0.51 3 ( 09) (e, f)
a: Mean ± SE.
b: Number of animals providing the data indicated in parenthesis.
c: Ten representative animals were necropsied in the control and 0.2% groups.
d: The kidneys were weighed with the adrenal glands attached.
e: Significantly different (p0.05) from the control group
f: One prostate was lost due to technical error.

	Table 4	Summary of Data from Sperm Studies (Task 2) Oxalic Acid
			Weight
Treatment	Body	R. Caudal	R. Epididymal	R. Testicular	Sperm Motility	Sperm Density	Abnormal Sperm
Group	(g)	(mg)	(mg)	(g)	(%)	x 10^6 a	(%)
Control	40.271 1.80 (10)b,c	16.980 10.778(10)	51.25011.912 (10)	0.1341 0.007 (10)	95.2 10.61 (10)	787 t 82(09)d	5.33 t 0.98(09)d
0,20%	44.841 2.10 (10)	17.750 10.725 (10)	50.77011.778 (10)	0.141 t 0.012 (10)	94.3 1 0.87 (10)	987 1103 (09)	7.20t 0.96(091
a: Per g caudal tissue.
b: Number of animals providing the data indicated In parenthesis.
c: Mean t SE.
d: Sperm suspensions from 2 animals (1 control and 1 treated) were accidentally mixed; the suspensions were discarded

Table 5.	Summary of Pup Survival and Body Weight Data (Task 2 - Final Litter) Oxalic Acid
		Treatment Oroup
	Parameter
		Control	0,10%	0,20%
NUMBER OF BREEDING PAIRS		40	19	20
NUMBER OF LITTERS BORN		38	19	18
TOTAL LIVE PUPS PER LITTER
Age	0	12.47 ± 0.60 (38)a.b	12.47 ± 1.10 (19)	11.78! 0.61 (18)
(Days)	4	11.37 ± 0.71 (38)	11.32 ± 1.21 ( 19)	10.78t 0.83 (18)
	14	11.09 ± 0.76(38)	11.26 ± 1.20 (19)	10.78± 0.83 (18)
LIVE MALE PUPS PER LITTER
Age	0	6.29± 0.36(38)	5.37 ± 0.61 ( 19)	5.56± 0.37 (18)
(Days)	4	5.58 ± 0.41 (38)	4.84 ± 0.64 (19)	5.17± 0.46 (18)
	14	5.47 ± 0.4308)	5.00 ± 0.69 (19)	5.11± 0.44 (18)
LIVE FEMALE PUPS PER LITTER
Age	0	6.18± 0.42 (38)	7.11 ± 0.70(19)	6.22± 0.50 (18)
(Days)	4	5.79± 0.46 (38)	6.47 ± 0.78 (19)	5.61± 0.51 (18)
	14	5.55± 0.47 (38)	6.26 ± 0.76 (19)	5.67± 0.52 (18)
LIVE MALE PUP WEIGHT (g)
Age	0	1.64± 0.03 (38)	1.64± 0.04 (18)	1.58± 0.03 ( 18)
(Days)	4	3.11± 0.09 (37)	3.17 ± 0.13 ( 17)	3.03± 0.09 (17)
	14	8.03± 0.26 (35)	7.64± 0.42 ( 17)	7.49± 0.31 ( 17)
LIVE FEMALE PUP WEIGHT (g)
Age	0	1.55± 0.02 (38)	1.58± 0.04 (18)	1.55± 0.02 (18)
(Days)	4	2.92± 0.11 (38)	3.09± 0.14 ( 17)	2.96± 0.08 (17)
	14	7.83± 0.27 (35)	7.55± 0.44 ( 17)	7.46± 0.27 ( 17)
LIVE COMBINED PUP WEIGHT (g)
Age	0	1.60± 0.02 ( 38)	1.60± 0.04 ( 18)	1.56± 0.02 ( 18)
(Days)	4	2.98± 0.10 (38)	3.12± 0.14 ( 17)	2.99± 0.08 ( 17)
	14	7.93± 0.26 (35)	7.56± 0.43 ( 17)	7.46± 0.29 ( 17)
a: Mean +/- SE.
b:Number of fertile pairs providing the data indicated in parenthesis.

Table 6. Reproductive Performance of Second Generation Fertile Pairs (Task 4) Oxalic Acid
	Treatment Group
Reproductive Parameter	Control Male X	0.20% Male X
	Control Female	0.20% Female
LIVE PUPS PER LITTER
Male	6.07± 0.50(15)b	5.39 ± 0.58 (18)
Female	6.13 ±0.70(15)	4.33 ± 0.57 (18) c
Combined	12 .20 ±0.61(15)	9.72 ± 0.65(18) c
PROPORTION OF PUPS BORN ALIVE	0.99 ±0.01(15)	0.98 ±0.01(18)
SEX OF PUPS BORN ALIVE
(MALES/TOTAL)	0.51 ± 0.04 ( 15)	0.56 ±0.04(18)
LIVE PUP WEIGHT (g)
Male	1.54 t 0.03(15)	1.62±0.04(18)
Female	1.50 ± 0.04,(15)	1.55 ± 0.05 (18)
Combined	1.52 ± 0.03 (15)	1.60 ±0.04(18)
ADJUSTED LIVE PUP WEIGHT (g)d
Male	1.59 ± 0.04(15)	1.59 ± 0.04(18)
Female	1.54 ± 0.04(15)	1.52 ± 0.04 ( 18)
Combined	1.56 ± 0.04(15)	1.56 ± 0.04 ( 18)
a: Mean ±SE.
b: Number of fertile pairs providing the data indicated in parenthesis.
c: Significantly different (p0.05) from the control group.
d: Means adjusted for total number of live and dead pups per litter by analysis of covariance.

Conclusions:

In conclusion, oxalic acid administered in drinking water at up to the 0.1% dose level does not affect the fertility in adult or second generation CD-1 mice. Significant reduction (p<0.05) was noted with respect to the number of litters per pair and adjusted live pup weights during Task 2-at the 0.2% dose level. During Task 4, the total number of live pups and the number of live female pups delivered by second generation breeding pairs were significantly less (p<0.05) than the corresponding control values. The prostate gland in animals exposed to 0.2% oxalic acid was smaller as evidenced by significantly reduced absolute and adjusted weights at necropsy. For second generation mice, adjusted kidney weight for female animals and absolute kidney weight for male mice were significantly increased (p<0.05). SMVCE studies indicated that prolonged oxalic acid treatment may interfere with the relative frequency of estrus as evidenced by the data from first generation mice. The incidence of abnormal sperm was almost doubled in second generation mice receiving 0.2% oxalic acid in drinking water. Since the increase in the percentage of abnormal sperm was noted in both treated second generation animals (Task 4) and adult mice (Task 2), it is possible that oxalic acid interferes with spermiogenesis.
In a read across approach, this conclusion is used in the assessment of iron oxalate properties.

Executive summary:

The National Toxicology Program (NTP) has developed a reproductive toxicity system designated "Fertility Assessment by Continuous Breeding" (FACB). Caesarean originated Barrier-sustained (COBS) CD-1 (ICR)BR outbred albino mice are used for the FACB study. It consists of four related tasks, not all of which are necessarily performed for a given compound.  

The study consisted of three successive tasks designed to determine the effects of oxalic acid on reproduction and fertility in CD-1 mice. The chemical was administered in the drinking water.Task 1, which is not analyzed statistically, was performed in order to select the doses for Task 2.In the second task,40males and 40females were randomly paired and received a vehicle control dose,19pairs received a low dose of 0.05%, 19 pairs received a mid range dose of 0.10%, and20 pairs received a high dose of    0.20%.The pairs were housed together for 98 days, followed by a 21 day segregation period to allow for delivery of the final litters. Because the overall response (affected fertility) during Task 2 wasnegative, Task 3 was not performed and Task 4was performed using second generation animals from the control and high dose groups only.

In Task 4, the reproductive performance of the control and high dose offspring from the final Task 2litters was evaluated. Group A consisted of 20 pairs of control males and females, and Group B consisted of 20 pairs of high dose level males and females. After a seven day cohabitation period, the pairs were separated and the females were allowed to deliver their litters.

At the conclusion of Tasks 1,2 and 4, experimental animals were necropsied: the liver, kidneys, testes, epididymis, prostate, and seminal vesicles with coagulating glands are weighed and fixed for histopathologic evaluation. In addition, vaginal smears are prepared for 7 consecutive days prior to necropsy to check the effect on the estrous cycle. For male mice, sperm-are studied in detail to evaluate the effect on sperm density, sperm motility, and sperm head morphology.

Exposure to oxalic acid produced no adverse effects on mating or fertility.In Task 2, treatment with 0.20% oxalic acid resulted in significant decreases in the average number of litters per fertile pair, unadjusted pup weight(males only) and adjusted pup weight. Adjusted prostate weight was significantly decreased(by21%)in high dose males, and adjusted kidney weight was increased (by 9%) in high dose females.

In Task 4, the only significant result found in the litter analysis was a decrease at the high dose level in the average number of live pups per litter. At necropsy, kidney weight was significantly increased in high dose males (by11%)and females(by9 %) .

In conclusion, oxalic acid administered in drinking water at up to the 0.1% dose level does not affect the fertility in adult or second generation CD-1 mice.

Justification for classification or non-classification

No reproductive effect related to the tet item was observed. These results are considered relevant and are used in a read across approach.