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Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Specific details on test material used for the study:
Sodium formate, formic acid.

RS-Freetext:
The intracellular formate concentration was more
significantly increased following treatment at pH 6.9
compared to the treatment at pH 7.4. Intracellular ATP was
not changed at pH 7.4, but at pH 6.9. Similarly, viability
was significantly decreased after 2 h at pH 6.9, but not at
pH 7.4.

===========================================================
                        Treatment
           ------------------------------------------------
Time       30 mM NaHCOO, pH 7.4   |  30 mM HCOOH, pH 6.9
-----------------------------------------------------------
           Formate         ATP    |  Formate           ATP
(h)     (µmol/mg protein)  (%)    | (µmol/mg protein)  (%)
-----------------------------------------------------------
0          3 +/- 0.5       100    |    3 +/- 0.5       100 
2         38 +/- 4         100    |   38 +/- 4          70
24        17 +/- 2         100    |   50 +/- 5          50
===========================================================

Conclusions:
CL-Freetext:
The authors concluded that cytotoxicity was associated with
the undissociated formic acid, rather than with formate.
Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Principles of method if other than guideline:
No test guideline available. Interaction with mitochondrial electron chain was examined. Endpoint adressed: cellular energy supply.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
not applicable
Details on results:
Formate inhibits cytochrome c oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome aa3. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.

The Ki for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.
Conclusions:
Formate is a moderate inhibitor of cytochrome c oxidase. The Ki is between 1 and 30 mM.
Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Induction of optical nerve toxicity in rodents following treatment with nitrous oxide, N2O
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
neurotoxicity
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
other: i.p. injection
Vehicle:
physiological saline
Remarks:
Doses / Concentrations:
4 g methanol/ kg bw
Basis:
actual ingested
Control animals:
yes, concurrent no treatment
Details on study design:
Folate levels, formate oxidation rates, and optical nerve function were measured in rats after receiving methanol. Rats were either non-pretreteated or had been pre-treated with nitrous oxide, N2O.
Examinations:
Hepatic folate levels, formate oxidation rates, and optical nerve function (electroretinogram (ERG) and flash-prooked cortical potential (FEP)).

1. Hepatic folate concentrations and formate metabolism

Nitrous oxide inhibits methionine synthetase in pretreated rats. Consequently, the hepatic tetrahydrofolate (THF) level and the rate of formate oxidation are both reduced to 50% compared to untreated rats. The reduced levels are comparable to those observed in monkeys and humans.

Species

Total hepatic folate

(nmole/g)

Hepatic tetrahydro-folate

(nmole/g)

Rate of formate oxidation

(mg/kg/hr)

Rat, untreated

26.9±3.3

14.2 ± 0.9

69 ± 1.6

Rat, N2O-treated

28.5 ± 1.2

8.5 ± 0.8

34 ± 1.0

Cynomolgus monkey

25.5 ± 0.5

8.1 ± 0.2

34 ± 2.0

Humans

15.8 ± 0.8

6.5 ± 0.3

no data

(data from earlier publications)


2. Blood formate levels and pH

Methanol intoxication of pretreated rats resulted in acidosis and blood formate levels which were comparable to those seen in intoxicated monkeys and humans. Blood formate concentration ranged between 8-15 mM for 30-40 hours in the treated rats. 
Similar blood formate concentrations over these time periods have been shown to produce ocular toxicity in monkeys and are associated with visual toxicity in human methanol intoxication.
 

 

Species

Blood Formate

(mM)

Blood pH

Rat, N2O-treated

16.1 ± 0.7

6.91 ± 0.06

Monkeys

11.4 ± 1.2

7.19 ± 0.02

Humans

19.3 ± 4.4

6.93 ± 0.02


(rat data: means ± SD from 6 rats; measurements 60 hours after initial
 dose.
other data: compiled from earlier publications)


3. Functional tests
Statistically significant changes were seen in both the retinal function (by ERG; electroretinogram) and the optical nerve integrity (by FEP; flash-evoked cortical potential) at 36 hours after the initial dose until
 the end of the experiment at 60 hours after initial dosing.


4. Histopathology
Retina from the methanol-intoxicated rat showed diffuse edema and vacuolation at the junction of the inner and outer segments of the photoreceptor cells, and in the retinal pigmented epithelial cells.
Mitochondrial cristae swelling was seen in the retinal pigmented epithelium cells and photoreceptors of intoxicated rats. Ultrastructural changes were much less pronounced in the optical nerve than in the retina.

 

Conclusions:
Nitrous oxide inhibits methionine synthetase in rats. This is associated with 50% reduced tetrahydrofolate levels and formate oxidation rate compared to untreated rats. Under these conditions methanol treatment results in acidosis, and morphological anf functional lesions of teh optical nerve and the retina, i.e. the same lesions that are seen in humans. Thus, an animal model for the methanol-induced retinal and optical nerve toxicity was established. Ingestion of formate salts by humans could lead to the same adverse effects.

Intoxicated rats showed tetrahydrofolate levels, formate oxidation rates, blood pH and blood formate levels that were comparable to those observed in intoxicated monkeys and humans.

Functional and morphological changes in the retina were more pronounced than in the optical nerve after methanol intoxication with formate blood levels ranging between 8-15 mM over a time period of 30-40 hours. Comparable formate levels were seen in intoxicated monkeys and humans.
Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Examination of blood formate levels and ocular morphology following methanol administartion and intravenous formate infusion into monkeys.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
neurotoxicity
Species:
monkey
Strain:
other: Macaca mulatta
Sex:
male
Route of administration:
other: i.v. infusion
Vehicle:
other: buffered aqueous solution
Details on exposure:
Four male rhesus monkeys (Macaca mulatta) received buffered Na-formate [(sodium formate: formic acid, 10:1 (0.5 M)] i.v. after a  loading of 1.25 mmol/kg bw (57.5 mg/kg) sodium formate, with a mean  infusion rate of 3.1 mEq/kg/h [142.6 mg/kg bw/h)]. 
Duration of treatment / exposure:
The animals were continuously infused for the whole study duration.
Frequency of treatment:
singly
Remarks:
Doses / Concentrations:
143 mg/kg bw/hour
Basis:
other: i.v. infusion
No. of animals per sex per dose:
4

1.     Blood formate concentrations 
The procedure produced no acidosis. Blood pHs were maintained between 7.4 and 7.6. After 10 h, all animals accumulated maximum formate in blood between 10 and 30 mEq/L (460 - 1380 mg/L).
 The maximum blood levels that were measured were as follows (Report, Table 1)


 

Blood formate (mg/L)

Time (h)

Clinical observations

 

 

 

Pupillary reflex

Fundus changes (a)

Animal 1

1560

39

No response

Mydriasis 8 mm

Moderate

Animal 2

1380

50

No response

Severe

Animal 3

920

41

Mydriasis 8 mm

Severe

Animal 4

550

25

normal

Moderate

 (a) = optic disc edema        


2.
 Ocular effects

Under these conditions, pupillary reflexes were rapidly altered, and in most animals no response to light was observed between 24 and 48 h.
 

Ophthalmology revealed marked optic disc edema (mainly in the prelaminar region, central portion of the proximal part of the optic nerve without significantly reaching to the distal part. The retina including the ganglion-cell layer was completely normal.

Conclusions:
Fomate blood levels in the range of 450 mg/L and above (≥ 10 mM) may cause irreversible ocular damage in monkeys if persisting for several hours. This result (≥ 10 mM) can be extrapolated to humans.
Executive summary:

Formate blood levels in the range of 450 mg/L and above (≥ 10 mM) may cause irreversible ocular damage in monkeys if persisting for several hours. This result can be extrapolated to humans.

 

Elevated blood formate levels were seen in monkeys receiving sodium form formate via i.v. infusion over several hours at a rate of 143 mg/kg bw/hour. The maximal levels ranged between 550 mg/L after 25 hours and 1560 mg/L after 40 hours. Moderate optical disk edema was seen in the animal with 550 mg/L, and severe effects were seen at 1000 mg/L and above.

These results with formate were completely consistent with those findings after methanol-intoxication. Formic acid and not formaldehyde (McMartin et al., 1979) has to be considered the causative agent for ocular damage within the methanol-intoxication syndrome, irrespective of acidosis. The mechanism of formate toxicity may be seen in the inhibition of oxidative phosphorylation by formate based on the on findings that this substance is an efficient inhibitor of cytochrome oxidase.

It should be noted that in an earlier publication (Martin-Amat et al. (1977); cited in the OECD SIDS) ocular effects were reported to occur in monkeys also at slightly lower blood formate levels (350 -550 mg/L, i.e.8 -12 mM). It was therefore concluded that clear signs of ocular damage may occur in monkeys at formate blood levels of approx. 450 mg/L an higher, i.e. ≥ 10 mM and above (OECD, 2004)

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
Examination of photoreceptor cell toxicity of formic acid and formate in  vitro.
GLP compliance:
not specified
Type of method:
in vitro
Endpoint addressed:
neurotoxicity
Specific details on test material used for the study:
Formic acid (30 mM, pH 6.9)
or
Sodium formate (30 mM, pH 7.4)
Details on exposure:
A photoreceptor cell line (661W cells) was exposed to either sodium  formate (30 mM, pH 7.4) or formic acid (30 mM, pH 6.9). 
Analytical verification of doses or concentrations:
not specified
Examinations:
Cellular formate concentrations, cellular ATP concentrations and cell viability were assessed at 2 h and 24 h after start of treatment.

Intracellular formate concentrations increased from basal concentrations of 3 +/- 0.5 µmoles formate/mg protein to 38 +/- 4 µmoles formate/mg protein following 2 hours of exposure to either formate or formic acid. However, by 24 h of exposure intracellular formate concentrations were significantly greater in cells exposed to formic acid than in cells exposed to sodium formate. (50 +/- 5 µmoles formate/mg protein vs 17 +/- 2 µmoles formate/mg protein). 


Intracellular ATP concentrations were significantly decreased in cells exposed to formic acid following 2 hr (70% of control) or 24 hr (50% of control) of exposure. ATP concentrations were not altered in cells exposed to sodium formate.
 


Significant decreases in cell viability as assessed by propidium iodide staining were also apparent at 2 h in formic acid exposed cultures, but
 not in sodium formate exposed cultures.

 

Conclusions:
According to the authors these data provide evidence for pH dependent differences in the cytotoxic actions of formate. They are consistent with studies showing that the undissociated formic acid is the active inhibitor of mitochondrial cytochome oxidase and that formic acid is only permeable through the inner mitochondrial membrane in its undissociated form.
Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Abstract.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Examination of photoreceptor cell toxicity of formic acid and formate in  vitro.
GLP compliance:
not specified
Type of method:
in vitro
Endpoint addressed:
neurotoxicity
Specific details on test material used for the study:
Formic acid (30 mM, pH 6.9)
or
Sodium formate (30 mM, pH 7.4)
Details on exposure:
A photoreceptor cell line (661W cells) was exposed to either sodium  formate (30 mM, pH 7.4) or formic acid (30 mM, pH 6.9). 
Analytical verification of doses or concentrations:
not specified
Examinations:
Cellular formate concentrations, cellular ATP concentrations and cell viability were assessed at 2 h and 24 h after start of treatment.

Intracellular formate concentrations increased from basal concentrations of 3 +/- 0.5 µmoles formate/mg protein to 38 +/- 4 µmoles formate/mg protein following 2 hours of exposure to either formate or formic acid. However, by 24 h of exposure intracellular formate concentrations were significantly greater in cells exposed to formic acid than in cells exposed to sodium formate. (50 +/- 5 µmoles formate/mg protein vs 17 +/- 2 µmoles formate/mg protein). 


Intracellular ATP concentrations were significantly decreased in cells exposed to formic acid following 2 hr (70% of control) or 24 hr (50% of control) of exposure. ATP concentrations were not altered in cells exposed to sodium formate.
 


Significant decreases in cell viability as assessed by propidium iodide staining were also apparent at 2 h in formic acid exposed cultures, but
 not in sodium formate exposed cultures.

 

Conclusions:
According to the authors these data provide evidence for pH dependent differences in the cytotoxic actions of formate. They are consistent with studies showing that the undissociated formic acid is the active inhibitor of mitochondrial cytochome oxidase and that formic acid is only permeable through the inner mitochondrial membrane in its undissociated form.
Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Principles of method if other than guideline:
No test guideline available. Interaction with mitochondrial electron chain was examined. Endpoint adressed: cellular energy supply.
GLP compliance:
not specified
Type of method:
in vitro
Details on results:
Formate is a moderate inhibitor of cytochrome c oxidase in vitro, the Ki is approx. 6 mM.
Conclusions:
Formate is a moderate inhibitor of cytochrome c oxidase. The Ki is approx. 6 mM.

Description of key information

Formate is a moderate respiratory chain inhibitor (Ki approx. 6 mM) which may be associated with ocular damage seen when elevated formate levels persist (approx. blod levels 7mM, min. 24 hours). The ocular damage is associated with low hepatic folate levels and, hence, slow  formate metabolism. It was demonstrated to occur in vitro, and in vivo in non-human primates (monkeys) and in pretreated rats receiving formate.

Additional information

Experimental animals metabolise formate more rapidly than humans and non-human primates, primarily due to higher hepatic folate levels. As a consequence, formate does not accumulate. It may, however, accumulate in humans and monkeys. Therefore, these species differences obscure potential adverse formate effects in experimental animals but may occur in humans.

After methanol poisoning, irreversible ocular toxicity (retina, optical nerve) is seen in humans but not in rodents, and it is attributable to folate. This was confirmed by experiments in vitro and in vivo where functional and morphological changes of the retina and the optical nerve were demonstrated with formic acid and sodium formate (Emmrich, 2002). Monkeys developed ocular lesions when plasma formate levels were increased over an extended period of time (>550 mg/L [approx. 12 mM], > 24 hours) by means of i.v. infusion of sodium formate (Martin-Amat, 1978). Additionally, a rat model was developed to demonstrate ocular toxicity in rats. Pretreatment with N2O significantly reduces the folate level in rats, and functional and morphological lesions are then obtained in rats receiving methanol or sodium formate (Eells, 2000). Formate-induced retinal toxicity in pretreated, methanol-intoxicated rats was recently demonstrated to occur at 2.6 mM formate after 24 hours (Seme, 1999). According to Hanzlik, blood formate must exceed 7 mM (i.e. 315 mg/L) for at least 24 hours to produce irreversible ocular damage to occur in humans.

Folate is a moderate inhibitor (Kiapprox 6 mM) of cytochrome c oxydase, i.e. it impairs respiratory chain and proper ATP supply of the affected cells, it favours generation of lactic acid and, hence, acidosis on a cellular level (Nicholls, 1976; Erecinska and Wilson, 1980). This pattern was seen in photoreceptor cells and optical nerve cells after in vitro or in vivo formate treatment. However, the exact mechanism of ocular toxicity is still not fully understood. It should be noted that folate treatment in vivo does not lead to acidosis, in contrast to methanol poisoning.