Calcium diformate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study. Justification of test substance: potassium diformate decomposes into one molecule of each formate and formic acid. Thus, potassium diformate gives two formate anions on a molar basis. Local effects of the formic acid formed can be neglected because these are not expected with neutral salts (sodium, potassium, ammonium, calcium formate). Read across can therefore be limited to systemic effects.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; absorption of TS and  reversibility of effects  over a 4 week treatment-free period.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium formate(1:2)

EC Number:

243-934-6

EC Name:

Potassium formate(1:2)

Cas Number:

20642-05-1

IUPAC Name:

potassium formate(1:2)

Constituent 2

Reference substance name:

potassium formate (1:2)

IUPAC Name:

potassium formate (1:2)

Details on test material:

- Name of test material (as cited in study report): Formi-LHS; Formi LHS-Super
- Molecular formula (if other than submission substance): C2H3O4 K
- Molecular weight (if other than submission substance): 130.14
- Substance type: double salt
- Physical state: white powder
- Analytical purity: not reported; information available at the sponsor
- Impurities (identity and concentrations):
- Storage condition of test material: in sealed container at room temperature (10-30°C) in the dark

Test animals

Species:

rat

Strain:

other: Crl:CDBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 166 to 208 g; females; 136 to 173 g.
- Housing: groups of 5 rats per cage
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):  routinely 19 to 25°C
- Humidity (%): 40 to 70%
- Air changes (per hr): minimum 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral exposure:

- Dosing: test substance was mixed into the diet.
- Diet: ground SQC Rat and Mouse Maintenance Diet No. 1, free access to diet and to tap water.
- Stability of TS in diet: at least 2 weeks
- Replacement of diet: regularly within 2 weeks
- Dose levels:
- Main study: 0, 600, 1200, 3000 mg/kg bw/day.
- Recovery study: 0 and 3000 mg/kg bw/day.
- Absorption study: 3000 mg/kg bw/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The enzymatic method "Formic Acid" by Boehringen Mannheim was used

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 600, 1200, 3000 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

Main study: 10 per sex and dose group.
Recovery study: 10 per sex of the control and the high dose groups.
Absorption study: 10 per sex of the high dose group.

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: Satellite group: 4 weeks

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Mortality, clinical signs: observed at least daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: recorded before first treatment, at weekly intervals and  before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment and in week 12
- Dose groups that were examined: all main-study animals were examined before treatment and  all control and high dose animals in week 12.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13 in main study, week 17 in recovery study
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13 in main study, week 17 in recovery study
- How many animals: week 13 in main study, week 17 in recovery study

URINALYSIS: Yes
- Time schedule for collection of urine: week 13 in main study, week 17 in recovery study
- Metabolism cages used for collection of urine: yes


OTHER:

ABSORPTION
0.5 ml blood samples were taken on Day 1 of feeding and during weeks 13  and 17 by tail vein  puncture. Plasma was separated and stored at below  -10°C until analysis of formate using the Formic Acid method by  Boehringer Mannheim.

GROSS PATHOLOGY

- All animals except those of the absorption study  were subjected to terminal necropsy after sacrifice. 
- Organ weights: adrenals, kidneys, liver, testes and epididymides were  weighed before fixation.

HISTOPATHOLOGY
Histopathology: samples of the following tissues were fixed in 10%  buffered formalin. Eyes were fixed in Davidson's fluid.

Adrenals, aorta#, brain, cecum, colon, duodenum, eyes, femur with marrow  and articular surface#, gross lesions, Harderian glands#*, head#, heart,  ileum, jejunum, kidneys, lacrimal glands#*, larynx, liver, lungs, mammary  (females)#, mandibular lymph nodes, mesenteric lymph nodes, muscle  quadriceps#, esophagus, optic nerves#, ovaries, pancreas, pituitary,  prostate#, rectum, salivary glands, sciatic nerves, seminal vesicles#,  skin#, spinal cord (cervical, lumbar, thoracic)#, spleen, sternum with  bone marrow, stomach, testes and epididymides, thymus, thyroids an  parathyroids, tongue#, urinary bladder, uterus, nasal turbinates#*,  nasopharynx#*, vagina#, Zymbal glands*#.

# = tissue retained in fixative but not examined. * = retained in situ with the head

All organs and tissues in the control and high dose group animals were  histopathologically examined following hematoxylin and eosin staining.  Additionally, kidneys, lungs and gross lesions from the intermediate  groups were examined. Following observations in the stomachs of high dose animals, the stomachs  of the low and intermediate dose group animals were also examined,  together with the stomachs from the animals at the end of the recovery  period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Absorption and recovery within 4 weeks

Statistics:

Body weight gains, necropsy body weight, hematology and clinical  chemistry variables were analyzed using ANOVA, separately for each sex.  Dunnett's multiple test was used for comparison between control and  treated group. A regression test was performed to determine whether there  was a linear relationship between increasing dose and response. Organ weights were analyzed using ANCOVA and Dunnett's test. 
All recovery study data were analyzed using ANCOVA.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related clinical observations or mortalities. Of the 3 mortalities observed (2 controls, 1 high dose female), necropsy  confirmed that all were associated with the orbital sinus blood sampling.

BODY WEIGHT AND WEIGHT GAIN
In males, there was a significant and dose-related reduction of body  weight gain. The overall difference from the controls (Weeks 0-13) was  statistically significant in all three groups (p<0.05, <0.01, and <0.001  in the low, intermediate, and high dose, resp.). In females, body weight gain was also reduced. Significance  was gained  only in the high dose group (p<0.01). 

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A slight dose-related reduction of food intake was noted in all male  groups. In females, a reduction was seen only in the high dose group. No  level of statistical significance was reached in any group. In the treatment-free recovery groups food intake was comparable to the  control rats.
For details of resulting doses see "Remarks on results". The authors argued that reduced body weight gain and food intake were probabl relate to reduced palability of the test diets.

OPHTHALMOSCOPIC EXAMINATION
No treatment related effects noted.

HAEMATOLOGY
There were small but statistically significant differences from the  controls (cf. table in "Remarks on results"). Fluctuations in  white blood cell counts were noted for both sexes in all groups with no  clear dose-relation or significant trends. Observed changes were not regarded to be of biological or toxicological relevance.

CLINICAL CHEMISTRY
There were small but statistically significant differences from the  controls  (cf. table in "Remarks on results"). Clotting times were unchanged. Observed changes were not regarded to be of biological or toxicological relevance.

URINALYSIS
Urinary parameters were similar to the controls in all groups except pH.  There was a trend towards an increased urinary pH (pH 8 or 9) in the  intermediate and high dose groups of both sexes at the end of the  treatment period. 

After the 4-wk treatment-free period the majority of the high dose rats  had a urine pH of 6.0 or 7.0.


ORGAN WEIGHTS
At the end of the treatment period, group mean adjusted kidney, liver,  testes and epididymides weights in males were similar to the controls, as  were the mean weights of kidneys and liver in females. Reduced adrenal  weights compared to controls were noted in males (not significant) and in  females (p<0.05).

At the end of the treatment-free period, the mean liver weight of the  high dose males was slightly higher (p<0.01) than in controls.

GROSS PATHOLOGY
No remarkable findings, except thickening of the stomach, usually  involving the limiting ridge, at the end of the 13-week treatment period.  The effect was more pronounced in males, probably due to the higher  concentration of the TS in the diet (cf. table in "Remarks on results").

The effect subsided within the 4-week recovery period.


HISTOPATHOLOGY: NON-NEOPLASTIC
There was no other finding than a dose-related increase in the severity  and incidence of squamous cell hyperplasia in the stomach. No squamous  cell hyperplasia was noted in any of the male and female controls. The  histopathological finding correlated with the thick stomach findings  during necropsy.

The effect was largely reversible within the 4-week treatment-free period.


OTHER FINDINGS

Absorption study
Formate plasma levels following 3000 mg/kg bw/day were below the LOD  (Level of Detection, i.e. 62.54 µg/ml) in all but few animals on Day 1.  In Week 13, mean plasma levels were increased and slightly above the LOD  in animals from both sexes in the early morning, consistent with the  nocturnal feeding habit, but decreased within few hours to below the LOD. 

In Week 17, formate levels were below  the LOD at the end of the 4-week  recovery period.  Thus, there was no accumulation of formate detectable in plasma over  time. 

======================================================
Examination      LOD          Males         Females              
at (time) (µg/ml)       (µg/ml)        (µg/ml) 
--------------------------------------------------------------------------------              
 62.54 
Day 1          
18:00 h                     < LOD           < LOD     
24:00 h                max. 88.3       
Day 2 
06:00 h                max. 114.4
15:00 h                    < LOD           < LOD 

Week 13 18:00 h                    < LOD           < LOD 
next day, 06:00 h      mean: 158         mean: 96.6
next day, 15:00 h          < LOD          < LOD

End of 4-week recovery period            < LOD           < LOD 
======================================================

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity; calcium diformate

Effect level:

3 000 other: mg/kg/day (calculated)

Sex:

male/female

Basis for effect level:

other: Read across from potassium diformate (1:2). No systemic toxicity or target organ

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity; potassium diformate

Effect level:

3 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No systemic toxicity or target organ

3

Dose descriptor:

NOAEL

Remarks:

local irritation; potassium diformate

Effect level:

< 600 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Local irritation. Squamous cell hyperplasia in the forestomach of male and female rats seen at all tested dose levels.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Consumption of test substance:
The consumption of test substance was close to the nominal dose levels.

Dose group	Nominal dose (mg/kg/day	Males	Females
Low	600	591	589
Intermediate	1200	1177	1178
High	3000	2879	2951

 

Hematology:

At Week 13: There were small but statistically significant differences from the controls (cf. table). Clotting times were unchanged.

Fluctuations in white blood cell counts were noted for both sexes in all groups with no clear dose-relation or significant trends.

	Males			Females
	Low	Intermediate	High	Low	Intermediate	High
RBC			Incr*
MCV			Decr***		Decr*	Decr***
MCH			Decr**			Decr*
MCHC						Incr**
Platelet			Incr***
WBC			Incr**

Levels of statistical significance: * = p<0.05; ** = p<0.01; *** = p<0.001

RBC   = red blood cell count                         MCV   = mean cell volume
MCH   = mean cell hemoglobin                   MCHC = mean cell hemoglobin concentration
WBC  = white blood cell count                     

Incr      = increase                                           Decr    = decrease

At Week 17: At the end of the 4-week recovery significant changes were only noted in females as tabulated below.

 

	Males			Females
	Low	Intermediate	High	Low	Intermediate	High
Hb						Incr*
RBC						Incr*
HK						Incr*

 

Hb      = hemoglobin             HK = hematocrit

Clinical chemistry:
Isolated fluctuations were noted among all groups without a clear trend. Statistically significant findings are listed below.

At week 13:

 

	Males			Females
	Low	Intermediate	High	Low	Intermediate	High
AST			Incrtrend
AP			Incrtrend
Total bilirubin						Decr*
Total protein						Decr*
K			Decr***
Ca						Decr*
Creatinine			Decr***	Incr.*		Decr*
Urea				Incr.*	Incr.*
Total cholesterol				Incr.*	Incr.*
Globulin						Decr*
Albumin/globulin ratio						Decr*

Levels of statistical significance:        * = p<0.05;     ** = p<0.01;     *** = p<0.001
AST                = aspartate aminotransferase
AP                 = alkaline phosphatase
Totbilirub       = total bilirubin
A/G ratio        = albumin/globulinratio

At Week 17 

At the end of the 4-week recovery significant changes were as tabulated below.

 

	Males			Females
	Low	Intermediate	High	Low	Intermediate	High
AST			Incr**
AP			Incr***			Incr**
Glucose			Decr**			Decr**

 

Urinalysis:
Urinary parameters were similar to the controls in all groups except pH. There was a trend towards an increased urinary pH (pH 8 or 9) in the intermediate and high dose groups of both sexes at the end of the treatment period. 

After the 4-wk treatment-free period the majority of the high dose rats had a urine pH of 6.0 or 7.0.

 

Necropsy
Table: local effects: incidences of thickening of the stomach

 

	Males			Females
	Low	Intermediate	High	Low	Intermediate	High
Week 13	1/10	3/10	7/10	0/10	2/10	5/10
Week 17	0/10	0/10	2/10	0/10	0/10	0/10

 

Organ weights
At the end of the treatment period, group mean adjusted kidney, liver, testes and epididymides weights in males were similar to the controls, as were the mean weights of kidneys and liver in females. Reduced adrenal weights compared to controls were noted in males (not significant) and in females (p<0.05).

At the end of the treatment-free period, the mean liver weight of the high dose males was slightly higher (p<0.01) than in controls.

Histopathology

Table: Incidence of squamous cell hyperplasia in the forestomach (local effect)

 

	Males			Females
	Low	Intermediate	High	Low	Intermediate	High
Week 13
n	10	10	10	10	10	10
Grade 0	6	1	0	6	2	0
Grade 1	2	6	4	4	6	5
Grade 2	2	3	5	0	2	3
Grade 3	0	0	1	0	0	2
Week 17
n			10			9
Grade 0			6			7
Grade 1			4			3
Grade 2
Grade 3

 

 

Applicant's summary and conclusion

Conclusions:

Local effects were seen in the stomach in both sexes in a dose-dependent manner at 1200 and at 3000 mg/kg bw/day. Clear systemic toxicity was not seen at any dose, including 3000 mg/kg bw/day, the highest dose tested. The NOAEL for systemic toxicity was therefore 3000 mg potassium diformate/kg bw/day in the rat, equivalent to 3000 mg calcium diformate/kg bw/day.

Executive summary:

The repeated dose of potassium diformate(1:2) was examined in a subchronic rat study according to the OECD guideline 408 and under GLP conditions. The guideline requirements were exceeded in that an absorption study and a recovery study were also included.

 A read across from potassium diformate is justified because potassium diformate decomposes in aqueous solution into one molecule of each formate and formic acid, and the potassium ion. Thus, potassium diformate gives two formate anions on a molar basis. Local effects of the formic acid formed can be neglected because these are not expected with neutral salts (sodium, potassium, ammonium, calcium formate). Read across can therefore be limited to systemic effects.

 

In the 13-week oral fed study potassium diformate (1:2) was administered in the diet at dose levels of 0, 600, 1200, and 3000 mg/kg bw/day to 10 rats/sex/dose. The doses were selected based on the results of a 7 day range finding study. Additional animals were used in satellite studies, i.e. a 4-week recovery study (0 and 3000 mg/kg bw/day, 10 rats/sex/dose), and in an absorption study (3000 mg/kg bw/day, 10 rats/sex/dose).

 

In the absorption study, formate blood levels were below the level of detection (LOD; 62.54 µg/mL) in most instances. Slightly increased levels in the range 66 to 158 µg/mL were occasionally seen in some animals few hours after the nocturnal food uptake, but these declined to below the detection limit within few hours. Blood levels were also below the LOD at the end of the 4-week recovery period.

These findings indicate that formate is rapidly metabolised in the rat, and that there was no accumulation over the 13-week treatment period.

 

Main study: no mortality or clinical signs of toxicity were seen. Male body weight gain was significantly decreased in all treated groups, whereas in females body weight reduction gained a level of significance only in the high dose group animals. A slightly reduced food consumption was seen in groups with reduced body weights. Test substance consumption was close to the nominal doses, i.e. 0, 590, 1180, and 2880 (males) and 2950 (females) mg/kg bw/day, respectively. Reduced body weight gain and food consumption resulted possibly from a reduced palability of the diet, and from the local irritation.

 

There were no effects in ophthalmology. No clear effects were seen in hematology and clinical chemistry. Some parameters gained a level of statistical significance, but this was of no biological or toxicological relevance. Organ weights of male and female rats were similar to those of the controls.

 

Pathology revealed no remarkable finding except from local irritation, evidenced by the observation of a thickening of the limiting ridge in the stomach in all male groups and in the female high dose group at the end of the 13-week period. Histopathology revealed a dose-related increase in the incidence and severity of squamous cell hyperplasia in the forestomach of male and female rats. This effect was largely reversible in the 4-week recovery group.

 

Overall, there was no overt systemic toxicity or target organ in this study. A dose-related body weight decrease was seen in all treated males and in the high dose female group, possibly as a result of the reduced palability of the diet. The small but statistically significant changes in hematology and clinical chemistry parameters in the high dose animals were not of toxicological significance, due to the small changes and the lack of target organ toxicity.

A specific target organ was the stomach, based on local  irritation in the forestomach which caused a dose-related thickening of the stomach at all dose levels, and was confirmed to be squamous cell hyperplasia. This was considered to be a response to minor irritation by the test substance, rather than a toxic effect (Covance, 1998).

 

 

Conclusion:

Overall, dietary administration of potassium diformate (1:2) to rats at 600, 1200, and 3000 mg/kg bw and day for 13 weeks did not produce any overt systemic toxicity. Minor local irritation had occurred in the forestomach of both sexes, with effects being seen in the males at all dose levels. Recovery was not complete in all animals after the 4-week treatment-free period.

 

The authors did not derive NOAEL values, but they may be considered as follows:

 

 

Local irritation: NOAEL below 600 mg/kg bw/day, based on the irritation of the forestomach and the squamous cell hyperplasia seen at 600 mg/kg bw and day in both sexes.

Systemic toxicity: NOAEL_{(rat, 90 day study)} 3000 mg Formi LHS/kg bw/day, as no adverse effect was seen at the highest dose tested. Taking stoichiometry and formula weights into consideration, this is equivalent to 3000 mg calcium diformate/kg bw/day.

 

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408).