Potassium hydrogen oxalate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

May 8, 1984 - January 15, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

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.

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)

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Oxalic acid

EC Number:

205-634-3

EC Name:

Oxalic acid

Cas Number:

144-62-7

IUPAC Name:

oxalic acid

Details on test material:

- 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%

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system 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")

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
no data

DIET PREPARATION
not relevant


VEHICLE
not relevant

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0,2% (high dose)
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0,1% (mid dose)
Basis:
nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0,05% (low dose)
Basis:
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
- Other:

Positive control:

No positive control was used

Examinations

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

OTHER:

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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)

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

<= 0.1 other: %

Sex:

male/female

Basis for effect level:

other: The fertility of the adult mice was not affected up to 0,1% dose level. A significant reduction was noted with respect to the numbers of litters per pair and adjusted live pup weights during Task 2 at the 0,2% dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

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

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

<= 0.1 other: %

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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, 40 males and 40 females were randomly paired and received a vehicle control dose, 19 pairs received a low dose of 0.05%, 19 pairs received a mid range dose of 0.10%, and 20 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 was negative, Task 3 was not performed and Task 4 was 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 2 litters 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 (by 21%) 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  (by 11%) and females(by 9 %) .

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.