Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
FACB protocol followed (the deviations are not considered to have a major influence on the relevancy of the results for the current endpoint)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Oxalic acid
- Molecular formula (if other than submission substance): C2H2O4
- Molecular weight (if other than submission substance): 90.038
- Analytical purity: 99.0 +/- 0.6 %
- Lot/batch No.: Alll679
- Impurities (identity and concentrations):
Chloride, 0.002%
Sulphate: 0.002%
Calcium: 0.001%
Nitrogen compounds: 0.001%
Heavy metals: 0.0005%
Iron: 0.005%
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston NY)
- Age at study initiation (weeks) : (P) x 11; (F1) x 0
- Weight at study initiation: (P) Males: no data g; Females: no data g; (F1) Males: no data g; Females: no data g
- Fasting period before study: no data
- Housing:
Task 2. Day -7 to 0: 2 per cage ( sexes separately); day 0 - 98 :1 pair per cage; day 98 - 119: individually
Task 3. Not performed
Task 4. after weaning F1 pups are housed 2 per cage (same sex & siblings); during cohabition not related males and females are housed 1 pair per cage. After cohabition animals are housed individually.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: quarantine 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: see table 2

(a description of the tasks is included in “Any other information on materials and methods")
Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 98 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The doses have been analytically verified by a titrimetric method.

Triplicate portions (10 mL) of each water sample received from EHRT, Inc., were transferred to individual 250-mL beakers and mixed with
10 mL of 10% sulfuric acid.
The prepared samples, standards, and blanks were individually titrated with ~0.05 N potassium permanganate solution, using the method of Fowler and Bright.

The outcomes of the different reports have been summarized in table 1 in the field "any other information on materials and methods"
Duration of treatment / exposure:
(P) Males: 7 days before mating.
(P) Females: 7 days before mating, 98 days during mating, 21 days during resulting pregnancies, 21 days through weaning of their F1 offspring.
(F1) Males: 105 days at weaning, during growth into adulthood, mating and production of an F2 generation, until weaning of the F2 generation.
(F1) Females: 101 days at weaning, during growth into adulthood, mating and production of an F2 generation, until weaning of the F2 generation.
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until 74 +/- 10 days after weaning.
- Selection of parents from F1 generation when pups were [...] days of age. no data
- Age at mating of the mated animals in the study: 95 +/- 10 days
Dose / conc.:
0.2 other: % (high dose)
Remarks:
Nominal in water
Dose / conc.:
0.1 other: % (mid dose)
Remarks:
Nominal in water
Dose / conc.:
0.05 other: % (low dose)
Remarks:
Nominal in water
No. of animals per sex per dose:
Control: 40 males and 40 females
0,2%: 20 males and 20 females
0,1%: 20 males and 20 females
0,05%: 20 males and 20 females
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The estimated maximum tolerated dose (MTD) and two lower dose levels are selected from a dose range finding study (Task 1) to conduct Task 2. These dose levels are chosen based on the Task 1 results. These treatment groups are referred to as 'High, Mid, and Low dose groups.
- Rationale for animal assignment (if not random): random
Positive control:
No positive control was used
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations:
Task 1: day 0, 7 and 14
Task 2: day -7, 0, 7, 28, 56, 84, 112
Task 4: first day of weaning, first day of cohabition and once a week thereafter.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Task 1: daily
Task 2: no data
Task 4: once a week starting the week after cohabition
Oestrous cyclicity (parental animals):
Task 2 & 4: vaginal smears are prepared for 7 consecutive days prior to necropsy to check the effect on the estrous cycle
Sperm parameters (parental animals):
Parameters examined in/P/F1 male parental generations:
testis weight, epididymis weight, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, sperm density
Litter observations:
STANDARDISATION OF LITTERS
Pups delivered during day 98 to 119 of the Task 2 study will be allowed to stay with their parents. At weaning (age 21 days), each pup within a litter were assigned a random number by a computer or from "Statistical Tables" by R.J. Rohlf and R.R. Sokal (W.H. Freeman Co., San Francisco). A minimum of two male and two female pups wiere saved per litter. Random numbers were then arranged in descending order (or ascending order), males and females


PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
[number and sex of pups, average live pup weight, proportion of pups born alive, number of live pups per litter

GROSS EXAMINATION OF DEAD PUPS:
no ; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After task 2 all surviving (P) animals are sacrificed
After task 4 all surviving (F1) animals are sacrificed
- Maternal animals: After task 2 all surviving (P) animals are sacrificed after weaning.
After task 4 all surviving (F1) animals are sacrificed

GROSS NECROPSY

Males:
Various male reproductive parameters, i.e., sperm motility, sperm density, and sperm morphology are evaluated according to the NTP-Sperm Morphology Vaginal Cytology Evaluation Protocol.

Specific details are provided in the
SOP manual (not included in report)
Body weight;
Liver weight;
Right testicular weight;
Ventral prostate weight (pair);
Seminal vesicle weight (pair including the coagulating glands); Right epididymal weight;
Left testis with attached epididymas will be excised but not weighed.
Right caudal epididymal weight.
Combined kidney (left and right) weight with adrenal glands attached.

Females:
Body weight;
Liver weight;
Combined kidney (left and right) weight with adrenal glands attached.
The reproductive tract is excised but not weighed.


HISTOPATHOLOGY / ORGAN WEIGHTS
The histopathology is not defined in the study report.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals are sacrificed; the F2 offspring are all sacrificed
- These animals were subjected to postmortem examinations macroscopic as follows:
sex
weight


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
No data

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
No data
Statistics:
The Kruskal-Wallis test, a nonparametric analog of the one-way analysis of variance, tests the hypothesis that dose group medians are equal (i.e. no treatment differences) against the alternative hypothesis that at least one dose group median does not equal the others. Jonckheere's test considers the more specific alternative hypothesis of a monotone trend, either increasing or 'decreasing, among dose groups. Pairwise comparisons between two dose groups were made using Wilcoxon's rank-sum test, which is equivalent to the Kruskal-Wallis test with two dose groups. P-values were obtained for the Kruskal-Wallis test using a large sample chi-square approximation whereas a large sample normal approximation was used for both the Jonckheere and Wilcoxon tests.


For data expressed as a proportion, such as number fertile/number cohabited, a chi-square test for equality of proportions was used to test the general alternative of unequal proportions, whereas a more specific Cochran-Armitage test was used to test for monotone increasing or decreasing trend among dose groups. Pairwise comparisons of proportions were performed using Fisher's exact test.

Since the number of pups in a litter may influence the average pup weight in a litter, a parametric analysis of covariance was used to test for dose group differences in average pup weight, after adjustment for average litter size. Pairwise comparisons were performed using a t-test.

Williams' test is a parametric multiple comparisons procedure designed to detect the lowest dose that differs significantly from the control when the response to treatment is expected to increase or decrease with dose level. With only two dose groups Williams' test is-equivalent to a t-test.

All tests of hypotheses are two-tailed, meaning that dose' groups which differ may have medians that are greater than or less than other dose groups with no specific direction specified beforehand.
Reproductive indices:
Fertility and Mating Indices

A Cochran-Armitage test was used to test for a dose related trend in fertility and mating indicies. Pairwise comparisons between control and dose groups were made using Fisher's exact test, for which the P-value represents the probability of a more extreme observation in the direction indicated by the data.
Offspring viability indices:
No data
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
Gross pathological findings:
no effects observed
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 (table 3)

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 (table 4)
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. (table 5)
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 (table 6)

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.
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 22, 2015 to March 20, 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
dentification Acido oxalico - STD
Appearance Fine White Crystal
Chemical Name Oxalic Acid Dihydrated
Batch Number 15000900
CAS No. 6153-56-6
Manufactured by OXAQUIM S.A.
FÁBRICA Y DEPARTAMENTO TÉCNICO
Polígono Industrial "Las Horcas" s/n
44600 Alcañiz (Teruel)
España
Teléfono: +34 978 83 31 13
Fax: +34 978 83 38 61
Supplied by OXAQUIM S.A.
C/ Gregal, 3 Urbanización "Parc Llevant"
43764 EL CATLLAR (Tarragona)
España
Teléfono: +34 977 65 38 98
Fax: +34 977 65 39 30
Purity (%) 99.6%
Molecular Weight 126.07 g/mol
Molecular Formula C2O4H2.2H2O
Manufacture Date May 21, 2015
Expiry Date May 20, 2017
Stability of the test item dilution Seven Days
Stability of the test item 1 year
Storage Conditions Room Temperature (20 to 30 oC)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sainath Agencies,
- Age at study initiation: 5- 7 Months
- Weight at study initiation: Minimum 2710.3 and Maximum 3792.6 gm
- Housing: Individually in EN130S Noryl (Tecniplast) cages (approximately 653 mm x 653 mm). After randomization, male and female were individually. During the mating phase, animals were housed on one male: one female basis within each dose group. After successful mating, the females were housed individually during gestation.
- Diet (e.g. ad libitum): Teklad Certified Global High Fiber Rabbit Diet (Lot No. 2031C-062816MA) from ENVIGO was provided ad libitum
- Water (e.g. ad libitum):Aquaguard filtered tap water was provided ad libitum
- Acclimation period:10 -19 days under laboratory conditions, after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 to 20.5 0C
- Humidity (%): 55.5 to 65.1 %,
- Air changes (per hr): air-conditioned with adequate (above 10) air changes per hour.
- Photoperiod (hrs dark / hrs light):light cycle of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: November 29, 2016 To:January 22, 2017
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Twice a Weekly
- Diet Preparation: Dietary admixtures were prepared using the powdered feed.
Test item was triturated and weighed into a tarred glass beaker using a suitable weighing balance, mixed with powdered feed to prepare the pre-mix at higher concentration. Pre-mix was used to prepare the low, intermediate and high dose concentrations. Required amount of pre-mix and powdered feed was mixed to achieve different dose concentrations in feed. Feed for the control animals were prepared similarly without addition of test item. The feed was prepared and homogenized with the help of Homogenizer before providing to the animals.
- Storage temperature of food: Room Temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated and the linearity response was found to be linear (r2 = 1.000) in the range of 10.12 to 500.16 mg. The applied concentration was found homogeneously distributed in the feed at concentrations 1,000 mg/L, 5,000 mg/L and 10,000 mg/L for the test item respectively. (i.e. % coefficient of variation (RSD) ranged in between 1.18 to 2.62). The dose concentration obtained in all the dose groups was in agreement with the target concentration (i.e. ranged between 92.072 % to 97.949 %)
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1: 1
- Length of cohabitation: Animals were paired on a one male: two female, within each dose group, for a period of fourteen days
Duration of treatment / exposure:
The dosing was commenced on day 6 of gestation (post mating) and continued till day 29 of gestation.
Frequency of treatment:
Daily
Duration of test:
30 days
Dose / conc.:
1 000 ppm
Remarks:
Low dose
Dose / conc.:
5 000 ppm
Remarks:
Intermediate dose
Dose / conc.:
10 000 ppm
Remarks:
High dose: For Pre-natal developmental toxicity study in Rabbit, the doses for main study (10000PPM) was selected based up on dose range finding study. In Dose Range finding study, up-to 10000 PPM there were no test item related changes in reproductive indices observed.
No. of animals per sex per dose:
24 females.
Control animals:
yes
yes, plain diet
Details on study design:
TEST SYSTEM

Test System Young Adult New Zealand White Rabbits
Scientific Name Oryctolagus cuniculus
Justification Recommended by the guideline
Source Sainath Agencies,
1-6-197/45/D, Bapujinagar, Musheerabad,
Hyderabad-48, India
Registration No. 282/PO/Bt/S/2000/CPCSEA
Total number of Groups 4
Age at start of mating 5-7 months
Total number of animals 60 Male* and 120 Female
Total number of animals used 10 Male and 20 Female (Dose Range Finding Study)
48* Male and 96 Female (Main Study)
(Females were nulliparous and non-pregnant)
*Note: Male animals were sent for sacrifice after confirmation of last mating.

Total number of animals per group 5 Mated female (Dose Range Finding Study)
24 Mated female (Main Study)
Body weight when treated Minimum 2710.3 and Maximum 3792.6 gm
Identification By unique cage number and individual animal numbers marked with an indelible marker p en on the ear. The animals were marked with permanent animal numbers before the start of test item administration and refreshed weekly thereafter. The animals were marked with the temporary animal numbers at start acclimatization. Fetuses were identified with tag.
Randomization Female were assigned to groups on confirmation of amting i.e. on ‘0’ day of gestation in se quence beginning with the group following the last group assigned on the previous day.
Acclimatization 10-19 days under laboratory conditions, after veterinary examination. Only animals without any visible signs of illness were used for the study.
RATIONALE FOR DOSE LEVEL SELECTION

A GLP Dose Range finding (DRF) study was carried out in New Zealand White Rabbit before commencement of the main study to confirm the high dose level for test item. A total of three groups (Low, Intermediate and High) consisting of 5 mated female were treated at dose levels of 1000, 5000 and 10000 PPM. Control group animals were treated with powdered feed alone. Dose levels for Main Study were selected based on Dose Range Finding Study observation and results.
Dose levels for main study selected for Low, Intermediate and High at dose levels were 1000, 5000 and 10000 PPM, respectively. Control group animals were treated with powdered feed alone.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
No clinical signs were observed in any of the female animals treated at control (0 PPM) Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: NA

BODY WEIGHT: Yes
No significant difference in the body weight and body weight gain (%) was observed in pregnant female animals treated at control (0 PPM), Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Feed consumption of pregnant female animals at (0PPM), Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups was not significantly different throughout the treatment period


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter

- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes:all per litter
- Head examinations: Yes: half per litter
Statistics:
• Data are summarized in tabular form. Statistical analysis was performed using statplus program. Values are given as mean ± standard deviation (SD).
• All the data were checked for Normality with Shapiro-Wilk W test.
• All the data were checked for Homogeneity with Bartlett Chi-Square test.
• Data were subjected to perform Analysis of Variance (ANOVA) for continuous data.
• Discontinuous data were subjected to nonparametric test (Mann-Whitney U-Test).
P ≤ 0.05 (5% level of significance) was considered to represent significance in the respective parameters, P > 0.05 was considered not Significant
Indices:
Pre–implantation loss (%)
(Number of Corpora Lutea - number of implantation sites) / Number of Corpora Lutea x 100
Post–implantation loss (%)
(Number of implantation sites - Total number of live foetuses) / Number of implantation sites x 100
Sex Ratio (% males)
Number of male foetuses (Day 0) / Total number of foetuses (Day 0) x 100
Variation Incidence (%)
Number of foetuses with variation/ Total Number of foetuses examined x 100
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference in the body weight and body weight gain (%) was observed in pregnant female animals treated at control (0 PPM), Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups in the DRF and main study. Howerever in main study marginal decrease in body weight gain percentage was observed on day 3 to day 30 gestation day in Low (1000 PPM), Intermediate (5000 PPM) and High dose (10000 PPM) groups compared to cotrol group.
The body weight changes observed are marginal and could not be attributed to the test item administartion and does not represent a change of any biological significance
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Feed consumption of pregnant female animals at (0PPM), Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups was not significantly different throughout the treatment period in dose range finding study except significant decrease in feed consumption was observed in intermediate (5000 PPM) and high dose (10000 PPM) group on day 27 og gestation period when compared with control (0 PPM).
In main study in females during gestation day 0 marginal increase in feed consumption was observed in low (1000 PPM) and intermediate dose (5000 PPM) compareed to control (0 PPM) animals. On day 6 marginal decrease in feed consumption was noticed in Low (1000 PPM) dose compared to control, whereas on day 9,12, 15 and day 24 of gestation significant decrease in feed was noticed low and intermediate dose. On day 18 and 21 decrease in feed consumption was observed in low dose group whereas on day 27 significant decrease in feed consumption was recorded in intermediate dose while compared to control group of animals. These changes were within the normal range with respect to age and sex of the animals. Hence, the changes in feed consumption were considered as biological variation
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant difference was observed in gravid uterine weight
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormality was observed in any of the female animals from control, low, intermediate and high dose groups in both dose range finding and main study.
No test item related a change in organ weights of the animals was observed in dose range finding
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
no significant difference was observed in the extent of pre-implantation loss and post-implantation loss in all treated groups
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant difference was observed in early and late resorption and number of live foetuses in all tretment groups
Early or late resorptions:
no effects observed
Description (incidence and severity):

No significant difference was observed in early and late resorption and number of live foetuses in all tretment groups
Parameter Group G1 G2 G3 G4
Dose (PPM) 0 1000 5000 10000
No. of Dams 21 20 20 21
No. of CL Mean 7.38 7.55 7.35 6.57
SD 2.20 2.37 2.76 1.72
No. of Implantation Mean 6.43 6.00 6.30 5.43
SD 2.01 2.18 2.81 1.54
No. of Foetuses Mean 5.00 4.83 4.96 4.38
SD 2.77 3.13 3.17 2.41
Early Resorption Mean 0.38 0.20 0.20 0.33
SD 0.67 0.52 0.52 0.73
Late Resorption Mean 0.05 0.00 0.10 0.10
SD 0.22 0.00 0.31 0.30
Pre implantation loss Mean 0.95 1.55 1.05 1.14
SD 1.20 1.19 1.23 1.06
% 12.72 20.79 16.36 16.61
Post implantation loss Mean 0.67 0.20 0.40 0.43
SD 0.97 0.52 0.68 0.75
% 11.13 6.96 3.83 10.87
Dam with resorption/s Number 8 3 5 7
Number of dams with abortions Number
0 0 0 0
Number of dams with early deliveries Number
0 0 0 0
Number of dams with stillbirths Number
0 0 0 0
Number of dams with dead foetuses Number
1 0 1 0
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Parameter Group G1 G2 G3 G4
Dose (PPM) 0 1000 5000 10000
No. of Dams 21 20 20 21
Total No. of Foetuses 122 116 120 105
Mean Litter Size 5.81 5.80 5.95 5.00
No. of Live Foetuses Number 120 116 119 105
Mean 5.71 5.80 5.67 5.00
SD 2.12 2.44 2.71 1.84
% 98.36 100.00 99.17 100.00
No. of Dead Foetuses Number 2 0 1 0
Mean 0.10 0.00 0.05 0.00
SD 0.44 0.00 0.22 0.00
% 1.64 0.00 0.83 0.00
No. of Live Male Foetuses Number 59 60 53 48
Mean 2.46 2.50 2.21 2.00
SD 1.74 2.15 1.74 1.35
% 49.17 51.72 44.54 45.71
No. of Live Female Foetuses Number 61 56 66 57
Mean 2.54 2.33 2.75 2.38
SD 1.86 1.66 2.09 1.69
% 50.83 48.28 55.46 54.29
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 10 000 ppm
Based on:
test mat.
Basis for effect level:
other: observed effects were not treatment related hence highest tested dose considered as NOAEL
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was observed in litter and foetus weight in low, intermediate and high dose groups in dose range finding study. However, in main study combined foetus body weight was decrease in high dose (10000 PPM) groups as compared to control (0 PPM). Whereas in female foetuses of High dose revelead decrese in body weight compare to control group of animals
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): in main study combined foetus body weight was decrease in high dose (10000 PPM) groups as compared to control (0 PPM). Whereas in female foetuses of High dose revelead decrese in body weight compare to control group of animals
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No significant difference was observed in the sex ratio of foetuses in low, intermediate and high dose groups
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External examination of foetuses revealed two small sized foetus in control, four in low dose, three in intermediate dose and two in high dose group.
No significant difference was observed in external findings in all treatment groups

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination of foetuses revealed incompletely ossified, not ossified, bipartite, bipartite thoracic vertebrae, not ossified lumbar vertebrae; not ossified or incompletely ossified sacral vertebrae; not ossified caudal vertebrae; not ossified, incompletely ossified and/or misshapen/irregular sternal centers; not ossified or incompletely ossified manubrium and/or xyphoid; waviness in control, low, intermediate and high dose groups. These variations were isolated and when expressed on a foetus/litter basis, it was found to be not statistically significant.
Type and distribution of variations noted during skeletal examination at the dose levels of 1000, 5000 an 10000 PPM did not indicate any test item-related effects
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral examination of foetuses revealed Lungs: Absent Azygos lobe in two foetuses of control and one in low and high dose group foetuses, respectively. Microphthalmia was observed in four foetuses of control and high dose each. Whereas two in intermediate and three foetuses in low dose group was observed. Brain: Dilated ventricles were observed in two foetuses of control and one each in intermediate and high dose groups.
No significant difference was observed in visceral findings in all treatment groups
Details on embryotoxic / teratogenic effects:
The oral (dietary) administration of oxalic acid to pregnant New Zealand white Rabbits from day 6 to day 29 of gestation at dose levels of 1000, 5000 and 10000 PPM resulted in no treatment-related maternal and embryofoetal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 10 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
other: observed effects were not treatment related hence highest tested dose considered as NOAEL
Key result
Abnormalities:
effects observed, non-treatment-related
Developmental effects observed:
no

Study No.: 5671

 

Study Title: Prenatal Developmental Toxicity Study of OXALIC ACID in Rabbits (Dietary)

1.        Number of pregnant and non-pregnant dams

GROUP

G1

G2

G3

G4

DOSE (PPM)

0

1000

5000

10000

No. of Pregnant females

21

20

20

21

No. of Non-Pregnant females

03

04

04

03

 

2.        Number of dams with abortions, early deliveries, stillbirths, resorptions, and/or dead foetuses

GROUP

G1

G2

G3

G4

DOSE (PPM)

0

1000

5000

10000

Number of dams with abortions

0

0

0

0

Number of dams with early deliveries

0

0

0

0

Number of dams with stillbirths

0

0

0

0

Number of dams with resorptions

8

3

5

7

Number of dams with dead foetuses

1

0

1

0

 

3.        Pre-and post-implantation loss, number and percent  Body weight, body weight change and gravid uterine weight, including optionally, body weight change corrected for gravid uterine weight

GROUP

G1

G2

G3

G4

DOSE (PPM)

0

1000

5000

10000

Pre implantation loss (Number)

0.95

1.55

1.05

1.14

Pre implantation loss (%)

12.72

20.79

16.36

16.61

Post implantation loss (Number)

0.67

0.20

0.40

0.43

Post implantation loss (%)

11.13

6.96

3.83

10.87

Gravid Uterine Weight (G)

376.0784

378.2618

361.4792

324.3361

 

 

 

 

 

 

Group

Body weights (G)

Gestation Day

0

3

6

9

12

15

18

21

24

27

29

30

1

2904.8

2937.8

2974.0

3012.0

3055.2

3102.2

3156.8

3214.2

3274.6

3342.2

3413.8

3431.1

2

2981.3

3005.4

3032.7

3063.4

3099.1

3138.9

3183.2

3230.4

3282.0

3337.7

3399.5

3415.0

3

2972.2

2996.0

3023.7

3056.3

3090.1

3129.8

3186.1

3223.6

3276.5

3333.2

3395.4

3411.1

4

2981.2

3004.6

3032.5

3063.6

3099.1

3138.5

3183.5

3232.0

3284.5

3338.0

3406.2

3422.3

 

 

Group

Body weight Gain (%)

Gestation Day

3

6

9

12

15

18

21

24

27

29

30

1

1.14

2.40

3.71

5.20

6.83

8.71

10.69

12.78

15.11

17.58

18.18

2

0.81

1.72

2.76

3.96

5.30

6.79

8.37

10.11

11.98

14.05

14.57

3

0.80

1.73

2.82

3.96

5.29

7.20

8.45

10.23

12.14

14.23

14.76

4

0.79

1.72

2.77

3.96

5.29

6.80

8.43

10.20

11.99

14.29

14.84

4.        Mean number and percent of live offspring  Mean foetal/pup body weight by sex and sexes combined

Parameter

Group

G1

G2

G3

G4

Dose (PPM)

0

1000

5000

10000

No. of Dams

21

20

20

21

Total No. of Foetuses

122

116

120

105

No. of Live Foetuses

Mean

5.71

5.80

5.95

5.00

Number

120

116

119

105

%

98.36

100.00

99.17

100.00

Foetus weight by sexes combined (G): Mean

44.693

45.678

43.687

41.132

Foetus Weight (G): Mean

Male

45.195

48.682

43.403

41.719

Female

44.200

42.458

43.907

40.618

 

 

 

 

 

5.        Number and percent of foetuses and litters with malformation (including runts) and/or variation as well as description and incidences of malformations and main variations (and/or retardation)

EXTERNAL FINDINGS

Group

G1

G2

G3

G4

Dose (PPM)

0

1000

5000

10000

No. of Litters Examined

21

20

20

21

No. of Litters affected

2

4

3

2

% Litters affected

9.52

20.00

15.00

9.52

No. of Foetuses Examined

120

116

119

105

Variation Incidence – Number (%)

No. of Foetus with Variations

Total Variations

2 (1.67)

4 (3.45)

3 (2.52)

2 (1.90)

Body small in size

2 (1.67)

4 (3.45)

3 (2.52)

2 (1.90)

 

 

 

VISCERAL FINDINGS

Group

G1

G2

G3

G4

Dose (PPM)

0

1000

5000

10000

No. of Litters Examined

21

20

20

21

No. of Litters affected

5

5

3

6

% Litters affected

23.81

25.00

15.00

28.57

No. of Foetuses Examined

120

116

119

105

Variation Incidence – Number (%)

No. of Foetus with Variations

Total Variations

8 (6.67)

5 (4.31)

3 (2.52)

6 (5.71)

Liver: Pale

0

1 (0.86)

0

0

Kidneys: Pale

0

1 (0.86)

0

0

Lungs: Absent Azygos lobe

2 (1.67)

1 (0.86)

0

1 (0.95)

Eyes: Microphthalmia

4 (3.33)

3 (2.59)

2 (1.68)

4 (3.81)

Brain: Dilated ventricles

2 (1.67)

0

1 (0.84)

1 (0.95)

 

 

 

 

 

 

 

 

 

 

 

 

 

SKELETAL FINDINGS

Group

G1

G2

G3

G4

Dose (PPM)

0

1000

5000

10000

No. of Litters Examined

21

20

20

21

No. of Litters affected

9

7

11

6

% Litters affected

42.86

35.00

55.00

28.57

No. of Foetuses Examined

120

116

119

105

Variation Incidence – Number (%)

Total Variations

13 (10.83)

7 (6.03)

16 (13.45)

12 (11.43)

Thoracic Vertebra

Hemicentric

 1 (0.83)

0

0

0

Caudal Vertebra

Bipartite

1 (0.83)

0

0

0

Sternal centers (2)

Misshapen/Irregular shaped

0

0

1 (0.84)

0

Ribs: Left

Small Size

0

0

1 (0.84)

0

Detached

0

0

1 (0.84)

1 (0.95)

Rudimentary

4 (3.33)

3 (2.59)

8 (6.72)

8 (7.62)

Incompletely ossified

1 (0.83)

0

0

0

Ribs: Right

Small Size

0

0

1 (0.84)

0

Not ossified

1 (0.83)

0

0

0

Incompletely ossified

1 (0.83)

0

0

0

Rudimentary

8 (6.67)

3 (2.59)

4 (3.36)

9 (8.57)

Bifid

0

0

1 (0.84)

0

Detached

0

1 (0.86)

1 (0.84)

0

Sternal centers (2)

Misshapen/Irregular shaped

0

0

1 (0.84)

0

Xyphoid

Bent

0

1 (0.86)

0

0

 

 

 

Conclusions:
The test item, OXALIC ACID was administered daily by dietary route to three treatment groups of twenty four New Zealand white Rabbits per group from day 6 to 29 day of gestation at dose levels of 1000, 5000 and 10000 PPM,. A control group of twenty four females were treated with dietary powder feed alone.
No mortality was observed in any of the treated animal throughout the scheduled treatment period.
No clinical signs were observed in any of the female animals treated at control (0 PPM), Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups.
No significant difference was observed in body weight, body weight gain (%) and feed consumption attributable to treatment in all treatment groups when compared with control group.
No treatment-related effects were observed in reproduction parameters such as corpora lutea count, early and late resorption and number of live and dead foetuses, pre-implantation loss, post-implantation loss as well as foetal sex ratio at any dose level treated at 1000, 5000 and 10000 PPM.
Litter weight and foetus body weight in both sexes were significantly not different all treatment groups.
The external and visceral variations observed were randomly distributed across the groups. Therefore these findings were considered as incidental findings and not test item-related.
Type and distribution of variations noted during skeletal examination at the dose levels of 1000, 5000 and 10000 PPM did not indicate any test item-related effects.
The oral (dietary) administration of OXALIC ACID to pregnant New Zealand white Rabbits from day 6 to day 29 of gestation at dose levels of 1000, 5000 and 10000 PPM resulted in no treatment-related maternal and embryo foetal toxicity.
Hence, the No Observed Adverse Effect Level (NOAEL) of test item OXALIC ACID for maternal and embryo foetal toxicity in New Zealand white Rabbits via oral (dietary) route was concluded to be the highest dose level employed i.e., 10000 PPM


Executive summary:

Study Title:Prenatal Developmental Toxicity Study of OXALIC ACID in Rabbits (Dietary)

This study was designed to investigate theeffects of OXALIC ACID on the pregnant female and on embryonic and foetal development when administered orally (dietary) daily to mated female Rabbits from day of implantation to one day prior to scheduled caesarean section (i.e. day 6 through day 29 post mating) including assessment of maternal effects as well as death, structural abnormalities (soft tissue and skeletal), or altered growth in the foetus.

The studyis compatible with the requirements of theOECD Guidelines for the Testing of Chemicals, Number 414 “PrenatalDevelopmental Toxicity Study”, Adopted by the Council on 22ndJanuary 2001.

In dose Range Finding (DRF) study was carried out before the main experiment to confirm the dose level of test item for main study. Three groups (Low, Intermediate and high) consisting of 5 pregnant Female Rabbits each were treated dailyfrom day 6 to day 29 of gestation at dose level of1000, 3000 and 10000 PPM. All animals were observed for mortality/viability and clinical signs of toxicity. Feed consumption and body weight were recorded during treatment period. On day 30thof gestation period all the animals were sent for necropsy and gross pathological examinations.

Dose Range Finding (DRF), no effects were observed in mortality/viability, clinical signs, feed consumption, body weight, gross pathological andvariations/malformations in foetuses subjected for external examination.Dose levels for main study were selected based on dose range finding study results. The doses selected for main study were 1000, 5000 and 10000 PPM.

The test item (formulated in powder feed) was administered daily by oral (dietary) to threetreatmentgroups oftwenty four mated female New Zealand White Rabbits per group from day 6 to day 29 of gestation at dose level of1000, 5000 and 10000 PPM. A control group comprised of 24 female Rabbits was administered powder feed alone.

The analytical method was validated and the linearity response was found to be linear(r2= 1.000)in the range of 10.12 to 500.16 mg. The applied concentration was found homogeneously distributed in the feed at concentrations 1,000 mg/L, 5,000 mg/L and 10,000 mg/L for the test item respectively. (i.e. % coefficient of variation (RSD) ranged in between 1.18 to 2.62). The dose concentration obtained in all the dose groups was in agreement with the target concentration (i.e. ranged between 92.072 % to 97.949 %).Details of the analysis of dose formulation are given inAPPENDIX I.

The mated females were observed twice daily for viability/mortality. Clinical signs were observed twice daily for first three days and once daily thereafter until sacrifice.

Animals were paired on a one male: two female, within each dose group, for a period of fourteen days. Each female was examined for the presence of a copulation plug in the vagina, vaginal mucus membrane and mating behavior. The presence vaginal plug (Presence of sperm), vaginal mucus membrane (swollen valva and congested mucus membrane and mating behavior were considered as positive evidence of mating (Day 0 of gestation).

Body weights were recorded ongestation day 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and day 30. Feed consumption was recorded on gestation day 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29.All surviving animals were sacrificed by intravenous injection of thiopentone sodium. On cesarean section uteri were removed, weighed and examined for the number of implantation sites, resorption and number of live and dead foetuses. The number of corpora lutea was counted on ovaries. The foetuses were removed from uterus, identified, weighed, sexed and evaluated for external malformation/variation. All the fetuses (alternating foetuses within the litter) were processed for skeletal alterations and for visceral (soft tissue) alterations.

Results

Analytics:

Analysis of stability, homogeneity and dose concentration of the prepared dose formulations was within acceptable levels.

Mortality:

No mortality was observed in any of the treated animal through out the scheduled treatment periodin main study.

Clinical signs:                                                                                                                        

No clinical signs were observed in any of the female animals treated at control (0PPM) Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups.

Body weight:

No significant difference in the body weight and body weight gain (%) was observed in pregnant female animals treated at control (0PPM), Low (1000 PPM), Intermediate (5000 PPM and High dose (10000 PPM) groups.

Feed Consumption:

No significant difference was observed in the feed consumption attributable to treatment in low, intermediate and high dose group compared with control group.

  

Mating Record:

All female animals of control, low, intermediate and high dose groups showed positive evidence of mating. At necropsy, three females each of control, high and and four females of low and intermediate dose group were found non-pregnant.

Necropsy:

Necropsy performed on 30thday of gestation, no test item-related macroscopic findings were observed in any of the animal in all treatment groups in dose range finding and main study.

No test item related microscopic findings were observed in treated animals ofdose range finding study.

All other histopathological findings observed in various tissues during evaluation of test item group animals were comparable with control group and were considered incidental. These changes can usually be considered to be species, age, gender, congenital, physiological or mode of death related and are covered in background historical data of pathology.

Examinationof Uterine Contents:

No significant difference was observed in the gravid uterine weight, corpora lutea count, in early and late resorption and number of live foetuses as well as in pre and post implantation losses between control and treated groups.

Foetus weight:

No significant difference was observed in foetus weight between control and treated groups.

Sex ratio:

Sex ratio was calculated as percent (%) male and there was no significant difference was observed in sex ratio between control and treated groups.

Examination of Foetuses:

ExternalandVisceral examination

No test-item related variations/malformations were observed in foetuses subjected for external and visceral (soft tissue) examination.

Skeletalvariations/malformations

The number of foetuses showing skeletal variation in treatment groups were comparable with control group. In general, incidences of skeletal variations were unaffected by test item treatment.

No test item related external, visceral and skeletal variations/malformations were observed in any of the treated group. Most of the observations recorded were minor abnormalities and considered as routine variables.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subchronic
Species:
rabbit

Justification for classification or non-classification