Dipotassium oxalate

Toxicity to reproduction

Administrative data

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:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

National Toxicology Program protocol. The protocol is in line with the OECD guideline 416 protocol. Deviations are not considered to alter the reliability of the results. The test item was administered in drinking water to mice; The reproductive toxicity of oxalic acid was evaluated according to the Fertility Assessment by Continuous Breeding (FACB) protocol

Deviations:

yes

Remarks:

This NTP study was conducted according to the FACB protocol. Beside a 14 day dose range finding study, a continuous breeding phase (119 days), and the effects on the F1 offspring were determined.

Principles of method if other than guideline:

- Principle of test: The reproductive toxicity of oxalic acid was evaluated according to the Fertility Assessment by Continuous Breeding (FACB) protocol
- Short description of test conditions: The FACB protocol is divided in four tasks: Task 1: Dos-range-finding study; Task 2: Continuous Breeding Phase; Task 3: Determination of affected sex and Task 4: Offspring assessment; Oxalic acid was administered in drinking water available ad libitum. The test chemical was mixed with water at 0.0 (control), 0.25, 0.5, 1.0, 2.5, and 5.0% dose levels for Task 1 and 0.0 (control), 0.05, 0.10, and 0.2%
dose levels for Task 2.
- Parameters analysed / observed: Task 1: Body weight, average intake of feed/drinking water, mortality, clinical and/or non-clinical symptoms of toxicity, incidence of dehydration
Task 2: Body weight, pups delivery, total number of pups and number alive, minimum number of dead and cannibalized pups, examination for external abnormalties, determination of sex, pups weight, mortality, amount of feed/drinking water with the test material consumed per animal
Task 3: body weight, pups delivered during task 3, evaluation of pups, mortality and health surveillance, amount of feed and drinking water consumed with the test article, vaginal smears, number of live and dead pups per litter, sex ratio, the affected sex will be necropsied and organ weights will be recorded
Task 4: body weight, pups delivered during task 4, evaluation of pups, mortality and health surveillance, amount of feed and drinking water with the test article consumed, vaginal smears, necropsy female and male mice, histopathology

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

ethandioic acid

Cas Number:

144-62-7

Molecular formula:

C2H2O4

IUPAC Name:

ethandioic acid

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

The CD-1 mouse was recommended by the Sponsor. This selection was based on the availability of a large historical data on reproductive toxicology and teratology.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY.
- Age at study initiation: Task 1: approx. 8 weeks
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: conditions were not decribed in this report, Task1: two mice of the same sex will be housed per cage Task 2: Seven days males and females were separated, then paired into treatment groups, 98 days of cohabitation and subsequently 21 days separated again.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: two to five weeks for Task 1 and Task 2, respectively

IN-LIFE DATES: From: To: See table 1 under 'Any other information on material and methods'

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh dosing formulations were made every 3 weeks or less. Representative formulations were analyzed and found to be within ±10% of theoretical values. Rats received treatment continuously from the beginning of Task 2 (from weaning for F, rats) until necropsy at the end of Task 3 or 4. Drinking water was changed at least twice.
each week.

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 (how): individually

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 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.
- Age at mating of the mated animals in the study: 95 ± 10 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.05 other: %

Remarks:

nominal in the drinking water (for Task 2)

Doses / concentrations 2

Dose / conc.:

0.1 other: %

Remarks:

nominal in the drinking water (for Task 2)

Doses / concentrations 3

Dose / conc.:

0.2 other: %

Remarks:

nominal in the drinking water (for Task 2 and 4)

No. of animals per sex per dose:

For Task 2:
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
For Task 4:
0.2%: 20 males and 20 females
control: 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.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: Task 2: On days -7, 0, 7, 28, 56, 84, and 112
Task 4: All experimental animals will be weighed on the first day of weaning, the first day of cohabitation, and once a week thereafter until sacrifice.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Task 2: The amount of water (X ) containing the test article consumed will be monitored during.the following periods:
(i) days -7 to 0
(ii) days 0 to 7
(iii) days 28 to 35
(iv) days 56 to 63
(v) days 84 to 91
(vi) days 112 to 119
Task 4: Amount of Feed/prinking water with the Test Article Consumed:
The amount of test article consumed will be monitored on a weekly basis beginning with the week after cohabitation and continued until sacrifice.

OTHER:
Task 4: Vaginal Smears: Vaginal smears will be prepared from mice who did
not deliver any pups. In case the fertility in female offspring is significantly affected, all animals will be smeared. Vaginal smears were prepared for 7 days prior to necropsy.


Pups peliyered During day 0 to 98:
All cages will be inspected at least twice a day to check for the presence of pups. After the mother has completed delivery, the pups will be counted, sexed, and weighed. The pups will then be sacrificed by decapitation. If a female has not completed delivery by evening health surveillance, the pups will be checked the next day and this day will be considered postnatal day 0.

Evaluation of Pups:
The day of delivery will be designated as postnatal day 0 and the appropriate
time of delivery will be recorded. After the dam has delivered and has cleaned her pups, she will be weighed and the weight will be recorded. The total number
of pups and number live, minimum number of dead and cannibalized pups will be noted. All pups will be 'examined for external abnormalities. Live pups
will be sexed. The sex of a pup will be determined externally by the
distance between the urogenital and anal openings (male distance is twice the female distance, usually 2 mm vs. 1 mm) and male perineum appears slightly swollen (site of future scrotal sacs). Within a litter, all males will be placed on the balance and the weight will be recorded in grams to two decimal places. The same procedure will be followed for female pups. Dead or cannibalized pups will not be sexed or weighed. A Kim-wipe will be placed on the balance to prevent chilling of pups. These data will be recorded on the appropriate data sheets.

Pups deliyered during day 99 to 119:
Pups delivered during the 21 days of the holding period will be allowed to remain with their mothers. The various parameters (weight, sex, and count) will, however, be evaluated as described above.

Oestrous cyclicity (parental animals):

Vaginal smears were prepared for 7 days prior to necropsy.

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
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD), pup weight.

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: All surviving (P) animals [after Task 2]. After Task 4 all F1 animals were sacrificed
- Maternal animals: All surviving (P) animals after the last litter of each generation was weaned. After Task 4 all F1 animals were sacrificed.

GROSS NECROPSY
- Gross necropsy in males consisted of 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.

In females the following parameters were examined:
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
Histopathology of various tissues will be conducted according to the Standard Operating Procedures described in Section XXXII and XXIII of the Standard Operating Procedures Manual. It must be added that only the specific tissue selected by the Project Officer will be examined. Since no results for histopathology were reported it can be considered that there were no severe alterations at gross necropsy. Organ weights were reported as indicated under gross necropsy.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrified by decapitation immediately after completed delivery and evaluation and all F2 offspring were sacrificed immediately after evaluation.

GROSS NECROPSY
- Gross necropsy consisted of external examinations to detect any abnormalty. The pups were sexed and weighted

HISTOPATHOLOGY / ORGAN WEIGTHS
For pups not selected as parental animals no histopathology was performed or organ weights were determined.

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 at-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 at-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 Indicies
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:

Not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Only 2 animals died during Task 2 cohabitation phase, 1 female each in the 0.05 and 0.1% dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Although no significant change was observed the female body weight gain was slightly decreased in the high dose group as compared to the control.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

A dose-related decrease in daily water consumption was noted at 0.1 and 0.2% dose levels. Decreased water consumption did not, however, result in any
significant symptoms of clinical toxicity.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect on the relative frequency of different estrous stages except for percent estrus (11% vs. 21% in the treatment group). Additional experiments are necessary to confirm the adverse effects of oxalic acid on the estrus phase.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The occurrence of abnormal sperm was slightly increased in the high dose group.

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

0.1 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

reproductive function (sperm measures)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The organ weight of the kidney adjusted for body weight was significantly increased in femal 0.2% mice (Task 4) at the end of the experimental time of Task 4.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Hard, brown mass attached to liver (1 cm) in one male of the 0.2% group (Task 4), and hard, brown lesion attached to end of seminal vesicles (1 cm) in one male of the 0.2% group.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.1 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased abnormal sperm, slight decrease in prostate gland weights of F1 animals, increased relative frequency of estrus in 0.2% group females, total number of live pups and number of live female pups wasdecreased,

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.2 other: %

System:

male reproductive system

Organ:

dorsolateral prostate gland

ventral prostate gland

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

presumably yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

275 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

no

Any other information on results incl. tables

Male Body and Organ Weights at Necropsy (Task 2) Oxalic Acid

Variable	Treatment group
	Control	0.2%
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)	0.141 ± 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 ± 0.513(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 (p<0.05) from the control group.

f: One prostate was lost due to technical error.

 

Summary of Data from Sperm Studies (Task 2) Oxalic Acid

treatment group	weight				Sperm Motility (%)	Sperm Density E+06	Abnormal sperm (%)
	Body (g)	R. Caudal (mg)	R. Epididymal (mg)	R. Testicular (g)
Control	40.27 ± 1.80 (10)^b,c	16.980 ± 0.778 (10)	51.250 ± 1.912 (10)	0.134 ± 0.007 (10)	95.2 ± 0.61 (10)	787 ± 82(09)^d	5.33 ± 0.98 (09)^d
0.2%	44.84 ± 2.10 (10)	17.750 ± 0.725(10)	50.770 ± 1.778 (10)	0.141 ± 0.012 (10)	94.3 ± 0.87 (10)	987 ± 103 (09)	7.20 ± 0.96 (09)

a: Per g caudal tisuue.

b: Number of animals providing the data indicated in parenthesis.

c: Mean ± SE.

d: Sperm suspensions from 2 animals (1 control and 1 treated) were accidentally mixed, the suspensions were discarded.

Summary of Pup Survival and Body Weight Data (Task 2 - Final Litter) Oxalic Acid

Parameter		Treatment group
		Control	0,1%	0,2%
Number of breeding pairs		40	19	20
Number of litters born		38	19	18
Total live pups per litter
Age (Days)	0	12.47 ± 0.60 (38)^a,b	12.47 ± 1.10 (19)	11.78 ± 0.61 (18)
	4	11.37 ± 0.71 (38)	11.32 ± 1.21 (19)	10.78 ± 0.83 (18)
	14	11.03 ± 0.76 (38)	11.26 ± 1.20 (19)	10.78 ± 0.83(18)
Live male pups per litter
Age (Days)	0	6.29 ± 0.36 (38)	5.37 ± 0.61 (19)	5.56 ± 0.37 (18)
	4	5.58 ± 0.41 (38)	4.84 ± 0.64 (19)	5.17 ± 0.46 (18)
	14	5.47 ± 0.43 (38)	5.00 ± 0.69 (19)	5.11 ± 0.44 (18)
Live female pups per litter
Age (Days)	0	6.18 ± 0.42 (38)	7.11 ± 0.70 (19)	6.22 ± 0.50 (18)
	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 (Days)	0	1.64 ± 0.03 (38)	1.64 ± 0.04 (18)	1.58 ± 0.03 (18)
	4	3.11 ± 0.09 (37)	3.17 ± 0.13 (18)	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 (Days)	0	1.55 ± 0.02 (38)	1.58 ± 0.04 (18)	1.55 ± 0.02 (18)
	4	2.92 ± 0.11 (38)	3.09 ± 0.14(18)	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 (Days)	0	1.60 ± 0.02 (38)	1.60 ± 0.04 (18)	1.56 ± 0.02 (18)
	4	2.98 ± 0.10 (38)	3.12 ± 0.14 (18)	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.

Reproductive Performance of Second Generation Fertile Pairs (Task 4) Oxalic Acid

Reproductive Parameter^a	Treatment group
	Control Male x Control Female	0.2% Male x 0.2% 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 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 ± 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 (p<0.05) from the control group.

d: Means adjusted for total number of live and dead pups per litter by analysis of covariance.

 

 

 

Applicant's summary and conclusion

Conclusions:

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-l 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 gene~ation mice receiving 0.2% oxalic acid in drinking water. Since the increase in the percentage of abnormal sperm was noted in 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 and is evaluating a new reproductive toxicity system designated nFertility Assessment by Continuous Breeding (FACB). Caesarean originated Barrier-sustained (COBS) CD-l (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 present study consists of three Tasks. Task 1: dose range-finding study. This study was not statistically analysed. Task 2: the continuous breeding phase. In this part 40 males and 40 females were randomly paired for the vehicle control group. 19 pairs received the low dose of 0.05%, 19 pairs received the mid dose of 0.1% and 20 pairs were administered the high dose of 0.2% oxalic acid. The pairs were housed together for 98 days and were then separated for 21 days to allow delivery. Under normal conditions, i.e. a positive response (affected fertility), Task 3 would be performed. However, the effect on fertility in the present study was only marginal at the highest dose, thus, the study was continued with Task 4: effects on offspring. Second generation animals (20 each group and sex) were mated each from the control and the highest dose group for seven days. Then the animals were separated and were allowed to deliver the litters.

At the end of all tasks (1, 2 and 4) the 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 at any dose group produced no adverse effects on mating or fertility. In Task 2, treatment with 0.2% 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 (by 21%) in high dose males, and adjusted kidney weight was increased (by 9%) in high dose females. The frequency of estrus was prolonged in the 0.2% female mice in Task 2 and to a lesser extent in Task 4.

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 (by 11%) and females (by 9 %). The amount of abnormal sperm was increased in the 0.2% males and the prostate gland was significantly decreased.

In conclusion, oxalic acid administered in drinking water at up to the 0.1% dose level corresponding to 162 mg/kg bw, does not affect the fertility in adult or second generation CD-1 mice.