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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Ophthalmological examinations, sensory activity, motor activity assessments not performed. Effects on male and female reproductive organs examined. Plasma levels determined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
; Ophthalmological examinations, sensory activity, motor activity assessments not performed. Effects on male and female reproductive organs examined. Plasma levels determined.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Formamide
EC Number:
200-842-0
EC Name:
Formamide
Cas Number:
75-12-7
Molecular formula:
CH3NO
IUPAC Name:
formamide
Details on test material:
Analytical purity: approx. 100%.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY).
- Age at study initiation: 6 weeks
- Housing: Caging: in groups of 5 male and female animals
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/-3°F
- Humidity (%): 50 +/-15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The dose formulations were prepared by mixing form-amide with deionized water to give the required concentration. Because the dose formulations were solutions, no homogeneity studies were performed. Stability studies of a 0.6 mg/mL dose formulation of a lot not used in the animal studies were performed using HPLC. Stability was confirmed for at least 50 days for dose formulations stored in the dark in glass containers under ambient and refrigerated conditions, and for at least 7 days under simulated animal room conditions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material analysis: The test material was examined using IR-, UV-/visible light spectroscopy, proton nuclear magnetic resonance spectroscopy, Thin layer-, Gas-, and High-Performance Liquid Chromatogrophy (i.e. TC, GC, HPLC).
During the 3-month studies, the dose formulations were analyzed at the beginning, midpoint, and end of the studies (Table I4 of report; attached to rat carcinogenicity study record ). All 15 dose formulations analyzed for rats and mice were within 10% of the target concentrations.
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 20, 40, 80, and 160 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
- core 90-day study (10 animals per sex and dose)
- pharmacokinetic study (5 animals per sex and dose)
- clinical pathology study (10 animals per sex and dose)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a preceding 14-day study
- Rationale for selecting satellite groups: examination of pharmacokinetics and clinical chemistry
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
(1) Clinical examinations, body weight determination: initially, weekly, and at the end of the studies.

(2) Clinical pathology:
Blood was collected from the retroorbital sinus of clinical pathology study rats on days 4 and 23 and from core study rats at the end of the studies for hematology and clinical chemistry.

Parameters:
Hematology: automated hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, nucleated erythrocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials.

Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.

(3) Histopathology:
Complete histopathology was performed on core study vehicle control and 160 mg/kg animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone (including marrow), brain, clitoral gland, esophagus, gallbladder (mice), heart (including aorta), large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lung (and mainstream bronchi), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular) testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. The epididymis and testis of male rats were examined in the remaining dosed groups.

(4) Sperm motility and vaginal cytology:
At the end of the studies, sperm samples were collected from core study males in the 0, 20, 40, and 80 mg/kg groups. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from core study females in the 0, 20, 40, and 80 mg/kg groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.

(5) Determinations of formamide in plasma: On days 9, 23, and 93, five male and five female plasma study rats were anesthetized with a CO2/O2 mixture, and blood was drawn from the retroorbital sinus for determination of formamide concentrations in plasma.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Determinations of formamide in plasma.
Sperm count (males) and estrous cycle (females).
Statistics:
The statistical analysis included:
Survival analysis: probability of survival was estimated according to Kaplan-Meyer (1958); dose-related effects on survival were analyzed using Cox's (1972) method.
Organ and body weight data: according to Dunnett (1955) and Williams (1971, 1972).
Clinical pathology, including hematology, clinical chemistry, sperm count and estrous cycle data: Shirley (1977), Dunn (1964), Jonckheere (1954).

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 efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clincl signs or mortalities in any in any group at 0, 10, 20, 40, 80, and 160 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN
The final body weight was statiscally significantly reduced compared to controls in males at 80 and 160 mg/kg bw/day and in females at 80, and 160 mg/kg bw/day. Terminal body weight was reduced by approx. 25% at 160 mg/kg bw/day, and by approx. 7% at 80 mg/kg bw/day (cf. table below).

HAEMATOLOGY
On day 23 and at week 14, there was a dose-related increase in the erythron, evidenced by increases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. On day 23, the erythron increase occurred in 80 and 160 mg/kg females (**) and 160 mg/kg males (**), but at week 14, groups administered 40 mg/kg or greater were also affected (**). The greatest magnitude increase occurred in hematocrit values (approximately 12%) and hemoglobin concentrations (approximately 15%) at week 14 in the 160 mg/kg groups. At week 14, the increase in the erythron was accompanied by increases in erythrocyte size, evidenced by 6% or less increases in mean cell volume in 40 mg/kg females(**) and 80 and 160 mg/kg males and females(**). The erythron increase was not accompanied by any alteration in reticulocyte counts. There were, apparent, albeit inconsistent, increases in nucleated erythrocyte counts in the 80 and 160 mg/kg groups. On day 23 and at week 14, there were apparent increases in segmented neutrophil counts in the 80 and 160 mg/kg groups that would suggest an inflammatory response or an alteration in the distribution of the neutrophils from the marginal pool to the circulating neutrophil pool. Platelet counts demonstrated small decreases in various higher dosed groups at all time points. The platelet count decreases are not considered to be clinically significant and may also indicate an alteration in peripheral distribution from circulating to marginal platelet pools. On day 23 (females) and at week 14 (males and females) there were small (10%) decreases in albumin and total protein concentrations in the 160 mg/kg groups that were probably related to the marked decrease in body weight gain for the 160 mg/kg groups. (* Significantly different (P=0.05) from the vehicle control group by Shirley’s test. ** P=0.01)

CLINICAL CHEMISTRY
No notable changes observed.

ORGAN WEIGHTS
No biologically significant organ weight changes were observed. Heart, kidneys, lung, liver, testis, thymus were weighed (report, Table G2).

HISTOPATHOLOGY: NON-NEOPLASTIC
The only microscopic findings attributed to treatment occurred in the testes and epididymis. There was minimal to mild degeneration of the germinal epithelium in the seminiferous tubules of the testes in seven 160 mg/kg males. This lesion was characterized by several changes including vacuolization, degeneration, and necrosis of spermatocytes and spermatids; disruption of the orderly appearance of the germinal epithelium; and retention of elongated spermatids. These lesions were observed in scattered individual or small clusters of seminiferous tubules. The epididymides of nine 160 mg/kg males contained a minimal increase above background levels of degenerate cells. These cells appeared to be degenerative (exfoliated) germinal epithelial cells (from the testes) often fused into multinucleated forms within the lumen of the epididymal tubules (see below for details).

OTHER FINDINGS:
- Formamide plasma levels:
The concentration of formamide in the plasma of male and female rats was monitored on days 9, 23, and 90 (cf. table below ). Plasma concentrations increased linearly with increasing dose at each time point (correlation coefficnt R²=0.9832 to 0.9997). However, at 40 mg/kg or greater there was a trend of increasing plasma concentration with duration of dosing.

- Reproductive organs/estrous cycle:
Reproductive organs:
Males: The number of spermatid heads per gram of cauda in 20 mg/kg males was significantly less than that of the vehicle controls, but this effect was not related to dose (cf. report, Table H1).
Females: females administered 40 or 80 mg/kg differed significantly from the vehicle controls in the relative time spent in the estrous stages. 40 mg/kg females spent more time in estrus and less time in diestrus than vehicle control females, but this effect was not relate dto dose (cf. report, Table H2).

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
40 - 80 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: based on the reduced body weight (-25%), erythron changes, and histopathological changes (degeneration of the germinal epithelium) in the testes and in the epididymis at 160 mg/kg bw
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: based on reduced body weights (-20%) at 160 mg/kg bw/day and hematological changes (increased erythron) at 80 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality:

There was no mortality in any of the male and female rats receiving 0, 10, 20, 40, 80, and 160 mg/kg bw/da

Body weight:

The final body weight was significantly reduced compared to controls in males at 80 and 160 mg/kg bw/day and in females at 80, and 160 mg/kg bw/day.

Table 1 : Body weight data (means)

  Mean Body Weight     

 Final Weight

         
 Dose (mg/kg)  Initial  Final Change Relative to Controls %          
 Male                  
 0 108±3  348±6  240±4  101          
 10 108±3  348±6  240±4  101          
 20 107±4  343±6  237±3  100          
 40 105±5  337±8  232±4   98          
 80 105±3   317±7**  213±5** 93           
 160 104±4   259±5**  155±5**   76          
                   
 Female                  
 0  92±3  188±3  96±3            
 10  91±3  191±4  100±3  101          
 20  92±3  184±3  92±3  98          
 40  90±3  177±3*  87±4  94          
 80  92±4  175±4*  83±3**  93          
 160  91±3  150±5**  59±3**  80          

(* Significantly different (P=0.05) from the vehicle control group by Williams' test. ** P=0.01)

Weights and weight changes are given as mean ± standard error

Reproductive organs:

Males: The number of spermatid heads per gram of cauda in 20 mg/kg males was significantly less than that of the vehicle controls; this effect was not related to dose (report, Table H1).

Table 2: Reproductive Tissue Evaluations for Male Rats (means)

   Vehicle control  20 mg/kg  40 mg/kg  80 mg/kg    

 n

 10  10  10  10    
 Weights (g)            
 Necropsy body weight  345±5  343±6  337±8  317±7*    
L. Cauda epidid.  0.166  0.180  0.173  0.174    
 L. Epididymis  0.454  0.485  0.475  0.470    
 L. Testis  1.558  1.606  1.601  1.523    
 spermatid measurements (1E07/g tissue)            
 Testis 148.1±5.6 153.1±8.5 144.7±5.8 149.6±6.3    
 Cauda 446.6±28.5 363.5±22.2*  440.2±17.1  433.4±23.9    
 Epididymal spermatozoal measurements            
 Motility (%)  81.14±1.24  79.58±0.81  77.92±0.53  79.01±1.11    

* Significantly different (P=0.05) from the control group by Dunnett’s test (body weights) or Dunn’s test (spermatid measurements)

Females:

Females administered 40 or 80 mg/kg differed significantly from the vehicle controls in the relative time spent in the estrous stages. 40 mg/kg females spent more time in estrus and less time in diestrus than vehicle control females (report, Table H2).

Table 3: Estrous Cycle Characterization for Female Rats (means)

   Vehicle Control  20 mg/kg  40 mg/kg  80 mg/kg    
 n  10  10  10  10    
 Necropsy body wt  188±3  184±3  177±3*  175±4**    
 Estrous cycle length (days)  4.80±0.25  4.80±0.11  5.25±0.34  5.00±0.00    
 Estrous stages (% of cycle)            
 Diestrus  65.0  59.2  50.8 60.0     
 Proestrus  10.0  9.2  10.8  15.0    
 Estrus  20.0  22.5  30.0  20.0    
 Metestrus  5.0  9.2  8.3 4.2     
 Uncertain diagnoses  0.0  0.0  0.0  0.8    

* Significantly different (P=0.05) from the control group by William’s test; **P=0.01

Histopathology

Table 4: Incidences of Selected Nonneoplastic Lesions in Male Rats

   Vehicle Control  10 mg/kg  20 mg/kg  40 mg/kg  80 mg/kg  160 mg/kg  
 Testes, n  10  10  10  10  10  10  
 Germinal Epithelium Degeneration              
 m  0  1  0  1  0  7**  
 Severity    2.0    1.0    1.3  
 Epididymis, n  10  10 10   10  10  10  
Germinal Epithelium Degeneration          
 m  0  1  0  0  0  9**  
 Severity    1.0        1.0  

** Significantly different (P=0.01) from the vehicle control group by the Fisher exact test

n = number of animals with tissue examined microscopically

m = number of animals with lesion & average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Pharmacokinetics:

Table 5: Formamide Concentrations (µg/mL) in Plasma of Rats (means)

   Control  10 mg/kg  20 mg/kg  40 mg/kg 80 mg/kg   160 mg/kg  
 Male (n)  5  5  5  5  5  5  
 Day 9  1.12  15.2**  31.2**  49.4**  113.5**  243.0**  
 Day 23  0.92  16.0**  33.4**  59.4**  147.8**  253.3**  
 Week 14  0.868  20.5**  39.9**  82.5**  182.2**  293.9**  
 Female              
 Day 9  0.66  15.9**  28.4**  58.2**  112.6**  240.4**  
 Day 23  1.84  17.8**  26.4**  49.7**  119.0**  223.2**  
 Week 14  1.2  19.5**  35.3**  76.7**  146.7** 298.0**   

* Significantly different (P=0.05) from the vehicle control group by Shirley’s test; ** P=0.01

Applicant's summary and conclusion

Executive summary:

Doses of formamide up to 160 mg/kg bw/day for 3 months produced a relatively mild toxic response in rats received 10, 20, 40, 80, and 160 mg/kg bw/day in a subchronic oral gavage study similar to OECD TG 408. Most effects were observed in groups that received the highest dose of 160 mg/kg bw/day and consisted of 20% to 25% body weight reductions and an increase in the erythron that was evidenced by increases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. The only microscopic lesion identified was degeneration of the germinal epithelium in the testes and epididymis of 160 mg/kg bw/day male rats. Slight body weight reductions also occurred in groups that received 80 mg/kg bw/day and in females that received 40 mg/kg bw/day; however, few other changes were noted at these doses.

The estimated split NOAEL values are 80 mg/kg bw/day for males, based on the reduced body weight (-25%), erythron changes, and histopathological changes (degeneration of the germinal epithelium) in the testes and in the epididymis, and 40 mg/kg bw/day for females, based on reduced body weights (-20%) at 160 mg/kg bw/day and hematological changes at 80 mg/kg bw/day (NTP, 2008).

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