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Toxicological information

Carcinogenicity

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Administrative data

Endpoint:
carcinogenicity: 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: Comparable to GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
Principles of method if other than guideline:
94/40/EEC. Amending Council Directive 87/153/EEC fixing guidelines for  the assessment of additives in animal nutrition.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): "Formi LHS-Super"
- Substance type: salt
- Physical state: powder
- Analytical purity: 98.44 and 99%.

Test animals

Species:
rat
Strain:
other: Crl:HanWist(Glx:BRL)BR
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Species: rat.
- Strain: Crl:HanWist(Glx:BRL)BR.
- Sex: male and female.
- Age: approx. 8 weeks at commencement of the experiment.
- Body weight: males: 186 to 275 g; females: 132 to 200 g.
- Number of animals: 20 per sex and dose group in the chronic toxicity study
- Housing: in groups of 5, in stainless steel mesh cages.
- Environment: routinely 19 to 25°C, relative humidity 40 to 70%, 
- Air changes: 15 air  changes/hour. 
- Photo period: 12-h light/dark cycle.

Treatment
- Dosing:  TS mixed into the diet.
- Diet: ground diet SQC Rat and Mouse Maintenance Diet No. 1, free access  to feed and tap water.
- Stability of TS in diet: examined; at least 7 days. 
- Dose levels: 0, 50, 400, 2000 mg/kg bw/day.
- Dose selection: based on results from preliminary studies including a  13-week study in rats

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING FEED:


DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): ground diet; mixed with TS in a mortar to give a premix, to which further diet was added as required and mixed in a blendor for five minutes.
- Storage temperature of food: at room temperatur ein sealed containers
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in week 1, and at 12 or 13 week intervals thereafter
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
7 day/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 400, 2000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a preceeding 13- week study (cf. Section 7.5)
- Rationale for selecting satellite groups: examination of repeated dose toxicity at week 52 (cf. Section 7.5)
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: recorded twice daily. Clinical signs: observed daily.
- Cage side observations were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:  Detailed physical examination of each  animal was performed at weekly intervals. 


BODY WEIGHT: Yes
- Time schedule for examinations: Body weight: recorded before first treatment, at weekly intervals for 16  weeks, once every 4 weeks thereafter 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
- Time interval:  determined weekly for the first 16 weeks, then one week  in every 4 weeks thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopy: in week 104, 20 animals/sex of the control and high dose groups were examined.
- Dose groups that were examined: control and high dose groups 


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 0.5 ml samples were withdrawn from 10 animals per group and sex in  Week 103. Blood was taken from the lateral caudal vein of fasted animals  and from decedents where possible.
- Animals fasted: Yes
- How many animals: 10 animals per group and sex 
- Parameters examined: hemoglobin concentration, packed cell volume, mean cell  volume, mean cell hemoglobin concentration, red blood cell count, total  and differential white blood cell count, mean cell hemoglobin, and  platelet count was performed and clotting times were determined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 0.5 ml samples were withdrawn from 10 animals per group and sex in  Week 103. Blood was taken from the lateral caudal vein of fasted animals  and from decedents where possible.
- Animals fasted: Yes
- How many animals: 10 animals per group and sex 
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline  phosphatase, sodium, potassium, calcium, chloride, inorganic phosphorus,  total protein, albumin, globulin, albumin/globulin ratio, total  cholesterol, glucose, urea, total bilirubin, creatinine.


URINALYSIS: Yes
- Time schedule for collection of urine: samples were collected from 10 animals per group and sex in Week  102.
- Animals fasted: yes. Where possible, urine and blood samples were taken from the same  animals.
- Prameters examined: microscopy of sediment, volume, specific gravity,  urobilinogen.  Semi-quantitatively: pH, protein, glucose, ketones, bilirubin, blood,  reducing substances.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Terminal examinations: all animals killed at termination or found dead  during the scheduled terminal necropsy period were considered to have completed the study, and any data collected was treated as such. All  animals including decedents were necropsied. Animals were weighed and  tissues were preserved. 
----------------------------------------------------------------------------------------------------------------------------
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 with mainstem bronchi#, mammary (females)#, mandibular 
lymph  nodes#, mesenteric lymph nodes#, muscle quadriceps, nasal turbinates*,  nasopharynx*, 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 and  parathyroids#, tissue masses#, tongue, 
trachea#, urinary bladder#,  uterus#, vagina#, Zymbal glands*.
----------------------------------------------------------------------------------------------------------------------------
# = microscopically examined * = retained in situ with the head

HISTOPATHOLOGY: Yes
Histopathology was performed on gross lesions, tissue masses and stomach  from all animals and tissues denoted by (#) in the tissue list from  control and high dose animals, and decedents from all groups. 


Statistics:
Statistical evaluation: Data from treated animals were compared with control data. 
In-life data: body weight gains, food consumption and hematology  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.
Survival data analysis: the Kaplan-Meier technique was used. Tests for  dose response in mortality were performed with the IARC annex (Peto et  al., 1980).  
Analysis of tumor data: the number of tumor bearing animals was analyzed  separately for males and females, for tumor types found in at least 3  animals of the given sex. Permutational tests were conducted in  accordance with the IARC annex (Peto et al., 1980).

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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:

CLINICAL SIGNS AND MORTALITY
Clinical signs: There were no clinical signs related to treatment.
Mortalities: The distribution of deaths at the end of Week 104 is tabulated below (Remarks on results). There was no pattern to indicate that the test substance had any effect on survival. Also, the incidence and pattern of morbidity observed in this strain was not changed by the test substance. The incidence of palpable masses was unchanged by treatment.


BODY WEIGHT AND WEIGHT GAIN
Compared to controls body weight and body weight gain was consistently lower in high dose animals, with gains over the 104 week period being significantly (p<0.001) lower in males (-27%) and females (-19%). There was no difference between control and low and mid dose animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption compared to controls was lower in the high dose males (-3%) and females (-6%; up to p<0.05). No effects were seen in the low and mid dose groups. Test substance consumption was generally close (within 1.5%) to the nominal target dose level. Cf. table below.

OPHTHALMOSCOPIC EXAMINATION
There was no effect on the eye noted

HAEMATOLOGY
There was no consistent pattern of variation to indicate an effect of treatment

CLINICAL CHEMISTRY
Urea levels for high dose males were consistently higher compared to controls at each of the investigations (p<0.01). Although in some instances variables attained levels of statistical significance compared to controls, there was no consistent pattern to indicate an effect of treatment.

URINALYSIS
There was no consistent pattern of variation to indicate an effect of treatment.

ORGAN WEIGHTS
There was no consistent pattern of variation to indicate an effect of treatment.

GROSS PATHOLOGY
In the stomach of high dose animals, there were dose-related increased incidences of nodules, raised focus and thick stomach when compared with controls. These correlated with microscopic findings. Cf. table below.
A decrease in subcutis masses was noted in high dose females. Other necropsy findings were similar in control and treated animals. There was no evidence of increased tissue masses suggestive of an oncogenic response.

HISTOPATHOLOGY: NON-NEOPLASTIC
The spectrum of microscopic findings was generally consistent with that expected for rats of this strain. Treatment-related changes were noted in the stomach, duodenum, salivary gland and kidney, probably due to the acidity of the test substance.

Compared to controls, findings in the stomach included (cf. table below under "Remarks on results):
- increased incidence and severity of basal cell/squamous cell hyperplasia at the limiting ridge in mid and high dose animals. This correlated with the macroscopic findings described above.
- acanthosis, hyperkeratosis, formation of variably sized and shaped papillae; associated with minor inflammatory cell infiltration in lamina propria and submucosa.
- foveolar epithelial hyperplasia in high dose animals.
- mild inflammatory lesions in the glandular stomach of high dose animals.
The NOEL was 50 mg/kg bw /day.

The scattered occurrence of other findings was similar to the spectrum expected for rats of this strain, or were considered to be a chance event, or to be related to the reduced food consumption/body weight, and were considered to be of no toxicological significance.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The spectrum of neoplasia was generally consistent with that expected in rats of this strain.
A reduced incidence of fibroadenoma in the mammary gland was noted in high dose females, probably related to the reduced food consumption/body weight.

There were no tumors of unusual nature or incidence indicative of specific target organ carcinogenicity in the stomach or any other tissue.
Relevance of carcinogenic effects / potential:
There was no evidence of a tumorigenic effect in the stomach or any other tissue.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No observation of local or systemic toxicity. TS Potassium diformate; corresponds to 17.7 mg formic acid/kg bw/day.
Remarks on result:
other:
Remarks:
Effect type: other: local toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Local toxicity: Histopathological changes in stomach of animals at 400 mg/kg bw/day. Systemic effects: Reduced food consumption and body weights at 2000 mg/kg bw/day. TS Potassium diformate; corresponds to 142 mg formic acid/kg bw/day.
Remarks on result:
other:
Remarks:
Effect type: other: systemic toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Absence of tumours or neoplastic lesions. TS Potassium diformate; corresponds to 708 mg formic acid/kg bw/day.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Any other information on results incl. tables

Analysis of formulations:

the target homogeneity of the test substance in the diet (85 to 115%) was sufficiently met (within +/- 10% of

nominal concentrations). Formulations were stable for up to 7 days at room temperature in the dark.


Mortalities:
The distribution of deaths at the end of Week 104 is tabulated below. There was no pattern to indicate that the

test substance had any effect  on survival.  

==============================================
Dose level         Males           Females   
(mg/kg bw/d)    [number of deaths (% survival)] 
----------------------------------------------
   0                 8 (84)          11 (78)
  50                12 (76)           9 (82)
 400                11 (78)          13 (74)  
2000                 7 (86)          12 (76)
==============================================


Test substance consumption: 
TS consumption was generally close (within 1.5%) to the nominal target  dose level:

==============================================
Target                Consumption of TS
Dose
                Males           Females   
(mg/kg bw/d)    (mg/kg bw/d)    (mg/kg bw/d) 
----------------------------------------------
  50               50.4                 50.6
 400              402.1                406.0  
2000             2012.6               2023.3
==============================================


Necropsy:

in the stomach of high dose animals, there were dose-related increased incidences of nodules, raised focus and thick

stomach when compared with controls. These correlated with microscopic findings.

Group incidence of findings in the stomach                        
===========================================================
              Males                      Females
Group        1M   2M   3M   4M        1F   2F   3F    4F

Dose group    0   50  400  2000 |      0   50  400   2000  

(mg/kg bw /d) 
-----------------------------------------------------------
n            8    12   12   7          11   11   16   12   

Thick        0     4    4   4           1    3    1    5   
Raised       0     0    1   0           0    0    0    0
focus
Nodule       0     0    0   0           0    0    0    2     
===========================================================
n = number of animals examined


  
Histopathology: 
Non-neoplastic observations


Group incidences of selected non-neoplastic microscopic findings  

============================================================
                               Males      |      Females   

------------------------------------------------------------
                           1M  2M  3M  4M  | 1F  2F   3F  4F 

Dose group  (mg/kg bw /d)  0  50  400 2000 | 0  50  400 2000  

------------------------------------------------------------
Stomach         n         42  38  39  43  | 39  39  36  38
basal/squamous   Grade 1    3   4  13   9  |  4   2  11   7
cell hyperplasia Grade 2    1   1   6  19  |  0   0   1  28  
                 Grade 3    0   0   0  14  |  0   0   0   3

foveolar epi-    Grade 1    1   3   3  17  |  0   0   0  21
thelium hyper-   Grade 2    0   0   0  23  |  0   0   0   0
plasia           Grade 3    0   0   0   1  |  0   0   0   0
  
glandular sto-   Grade 1    3   1   1   7  |  0   3   1   6
mach gastritis   Grade 2    0   0   1   0  |  0   0   0   0  
   
Duodenum         n         42   1   5  43  | 39   3   3  38
Brunner's gland  Incidence  0   0   0  16  |  0   0   0   8
hypertrophy  

Salivary gland   n         42   1   0  43  | 39   0   0  38
acinar cell      Incidence  0   0      17  |  0          10
hypertrophy

Kidney           n         42   3   7  43  | 39   8   0  38 
pelvic mineral. Incidence  28   3   3   4  | 37   6      20
papillary mineral. Inc.     1   1   1   1  |  8   1       2
pyelitis        Incidence  15   2   4   0  |  8   2       4
============================================================
n = number of animals examined
Grade 1, 2, 3: minimal, slight, moderate, respectively.
mineral. = mineralization


 

Applicant's summary and conclusion

Executive summary:

Conclusion:

In a combined toxicity/carcinogenicity study that was equivalent to an OECD guideline No. 453 and conducted und GLP conditions, the oral administration of potassium diformate (1:2) to rats (50/sex/dose) at dose levels of 50, 400, and 2000 mg/kg bw/day was well tolerated including the top dose without effects on clinical condition or survival. Depression of food consumption and body weight with sequel was observed in rats at 2000 mg/kg bw/day. The NOAEL for systemic toxicity was 400 mg potassium diformate/kg bw/day.

Adaptive hyperplastic changes in the stomach and the gastro-intestinal tract were seen in rats at 400 and, to a higher extend, at 2000 mg/kg bw/day.The NOEL was 50 mg potassium diformate/kg bw/day for these effects.

There was no evidence of a tumorigenic effect in the stomach or any other tissue, i.e. the NOAEL for carcinogenic effects was 2000 mg potassium diformate/kg bw/day.

Taking the molecular weights of potassium diformate (130.1) and formic acid (46.03) into consideration, the following dose descriptors are calculated for formic acid from the above figures using a multiplier of 0.354 (46/136 = 0.354):

 

NOEL gastro-intestinal changes:         17.7 mg formic acid/ kg bw/day

NOAEL systemic toxicity:                    142 mg formic acid/ kg bw/day

NOAEL carcinogenicity:                      708 mg formic acid/ kg bw/day