Registration Dossier

Endpoint conclusion:

no study available

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: limited pathology and blood work undertaken

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

limited pathology and blood work undertaken

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

Swiss

Sex:

male/female

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 wks

Frequency of treatment:

4h/d, 5d/wk

Remarks:

Doses / Concentrations:
0, 0.5, 5, 50%
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/gp

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEC

Effect level:

ca. 50 000 ppm

Sex:

male/female

Basis for effect level:

other: statistically significant reductions in body weight gains

Critical effects observed:

not specified

OBSERVATIONS:

Clinical signs of toxicity:

All animals survived to the scheduled necropsy.

Bodyweight and bodyweight gain:

Treatment related decreases in body weight were observed in high dose group animals, with a depression of 77 and 63% in body weight gain in males and females respectively. This depression in weight gain was statistically significant (p<0.025 and p<0.01, respectively).

Haematology & clinical chemistry:

No treatment related changes were observed in any of the parameter measured.

Urinalysis:

None undertaken

Sacrifice and Gross Pathology:

No treatment related changes were observed in any of the parameter measured.

Organ weights:

No discussion of organ weight data.

Conclusions:

The objective of the study was to demonstrate the maximum tolerated concentration of N2O, which was deemed to be 50% (500000 ppm). In terms of establishing a NOAEL, based on the results of this study and the data presented, 50000 ppm (5%) was deemed to be the NOAEL, based on statistically significant reductions in body weight gains observed at the LOAEL (500000 ppm [50%]).

Executive summary:

Swiss Webster mice (15/sex/gp) were exposed to N2Oviawhole body inhalation at concentrations of 0, 5000, 50000 or 500000 ppm [0, 0.5, 5, 50%] for 4/h/d, 5d/wk over 14 wks. At necropsy limited histopathology and haematology / biochemistry parameters were measured.

All animals survived to the scheduled necropsy. The study failed to demonstrate exposure related haematopoietic changes. There was no change in the white blood cell count nor was granulocytopenia or thrombocytopenia observed. The lack of effect suggests that either the strain of mouse was insensitive to N2O, or more likely that continuous exposure is necessary to induce leucocytopenia, as previous demonstrated following continuous exposure to N2O at high concentrations (20-80%). Furthermore, no treatment related changes in organ weights, biochemical or histopathological parameters were observed.

Treatment related decreases in body weight were observed in high dose group animals, with a depression of 77 and 63% in body weight gain in males and females respectively. This depression in weight gain was statistically significant (p<0.025 and p<0.01, respectively).

The objective of the study was to demonstrate the maximum tolerated concentration of N2O, which was deemed to be 50% (500000 ppm). In terms of establishing a NOAEL, based on the results of this study and the data presented, 50000 ppm (5%) was deemed to be the NOAEL, based on statistically significant reductions in body weight gains observed at the LOAEL (500000 ppm [50%]).

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

91 500 mg/m³

Study duration:

subchronic

Species:

mouse

Quality of whole database:

KL. 2/3 limited pathology and blood work undertaken

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: limited pathology and blood work undertaken

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

limited pathology and blood work undertaken

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

Swiss

Sex:

male/female

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 wks

Frequency of treatment:

4h/d, 5d/wk

Remarks:

Doses / Concentrations:
0, 0.5, 5, 50%
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/gp

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEC

Effect level:

ca. 50 000 ppm

Sex:

male/female

Basis for effect level:

other: statistically significant reductions in body weight gains

Critical effects observed:

not specified

OBSERVATIONS:

Clinical signs of toxicity:

All animals survived to the scheduled necropsy.

Bodyweight and bodyweight gain:

Treatment related decreases in body weight were observed in high dose group animals, with a depression of 77 and 63% in body weight gain in males and females respectively. This depression in weight gain was statistically significant (p<0.025 and p<0.01, respectively).

Haematology & clinical chemistry:

No treatment related changes were observed in any of the parameter measured.

Urinalysis:

None undertaken

Sacrifice and Gross Pathology:

No treatment related changes were observed in any of the parameter measured.

Organ weights:

No discussion of organ weight data.

Conclusions:

The objective of the study was to demonstrate the maximum tolerated concentration of N2O, which was deemed to be 50% (500000 ppm). In terms of establishing a NOAEL, based on the results of this study and the data presented, 50000 ppm (5%) was deemed to be the NOAEL, based on statistically significant reductions in body weight gains observed at the LOAEL (500000 ppm [50%]).

Executive summary:

Swiss Webster mice (15/sex/gp) were exposed to N2Oviawhole body inhalation at concentrations of 0, 5000, 50000 or 500000 ppm [0, 0.5, 5, 50%] for 4/h/d, 5d/wk over 14 wks. At necropsy limited histopathology and haematology / biochemistry parameters were measured.

All animals survived to the scheduled necropsy. The study failed to demonstrate exposure related haematopoietic changes. There was no change in the white blood cell count nor was granulocytopenia or thrombocytopenia observed. The lack of effect suggests that either the strain of mouse was insensitive to N2O, or more likely that continuous exposure is necessary to induce leucocytopenia, as previous demonstrated following continuous exposure to N2O at high concentrations (20-80%). Furthermore, no treatment related changes in organ weights, biochemical or histopathological parameters were observed.

Treatment related decreases in body weight were observed in high dose group animals, with a depression of 77 and 63% in body weight gain in males and females respectively. This depression in weight gain was statistically significant (p<0.025 and p<0.01, respectively).

The objective of the study was to demonstrate the maximum tolerated concentration of N2O, which was deemed to be 50% (500000 ppm). In terms of establishing a NOAEL, based on the results of this study and the data presented, 50000 ppm (5%) was deemed to be the NOAEL, based on statistically significant reductions in body weight gains observed at the LOAEL (500000 ppm [50%]).

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

915 000 mg/m³

Study duration:

subchronic

Species:

mouse

Quality of whole database:

KL. 2/3 limited pathology and blood work undertaken

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Repeat oral toxicity

As dinitrogen oxide is a gas, the oral route of exposure is not relevant.

Repeat dermal toxicity

As dinitrogen oxideis a gas, the dermal route of exposure is not relevant.

Repeat inhalationtoxicity

In a sub-chronic rat study (Riceet al.,1985) test material related changed were manifest as reductions in body weight gains in both males and females following exposure to N2O 4h/d, 5d/wk for 14 wks.. The study failed to demonstrate exposure related haematopoietic changes. There was no change in the white blood cell count nor was granulocytopenia or thrombocytopenia observed. Whilst no NOAEC value was set by the authors of the study (as this was not the objective), from the results and data presented an NOAEC of 5% (50000 ppm) could be deemed as the NOAEC, based on reductions in body weight gain (at the LOAEC of 50% [500000 ppm]).

In a 5 week study (Stevens et al.,1977) rats and mice were continuously exposed to N2O via whole body inhalation at a single dose level of 20%. The purpose of this study was not to establish a NOAEC, but rather to compare different anaesthetics. Limited histopathology was undertaken with only mice showing a greater incidence of focal inflammatory lesions of the liver, which consisted of portal infiltrates and midzonal granulomas. No hepatic necrosis was demonstrated. Rats showed no adverse histopathological changes related to N2O exposure.

Finally, in a study which the objective was to determine the tolerance of N2O (via abolishment of the righting reflex in 50% of animals), mice were continuously exposed to N2O via whole body inhalation for 3 weeks. Limited haematology and clinical chemistry analysis was undertaken but no histopathology. An increase in the synaptic membrane RBC membrane cholesterol/phospholipid ratios of N2O tolerant mice were seen. The biological relevance of this was not discussed.

In terms of enzymatic activities no differences were observed. Haemolysis studies failed to show any difference, confirming treated RBC from mice were no more fragile than control mice RBC. No detectable difference in the EPR spectra of spin labelled synaptic membranes from treated animals were observed.

In a review of N2O toxicity by Eger II (1985) the effects of this anaesthetic agent upon several different organ systems were discussed. N2O exerts many of its effects through interference of methionine synthase activity. N2O irreversibly oxidises vitamin B12, blocking the folate cycle. The consequence of these effects is directly comparable to the effects of vit B12 deficiency. In the liver, anaesthetic levels of N2O decrease in methionine synthase activity in rats, mice and humans. The decreases become greater with an increase in the duration of exposure. Any hepatotoxicity of N2O does not appear to result from an effect on cytochrome P450. The activity of Cyp P450 dependent reaction does not change in the livers of rats exposed to 75% N2O. This data contradict the effects seen in mice (Stevenset al., 1977) which suggest that the strain and species of mice used were vulnerable to the effects of N2O.

The hypoxic rat model has been used to test the hepatotoxicity of N2O. Rats pretreated with phenobarbital inhaled 92.5% (1/3 MAC) N2O for 46 minutes. The vulnerability of these animals to N2O-indued liver injury was greater than it was to 1/3 MAC enflurane, isoflurane or thiopental (given with 7.5% O₂).

Cardiovascular effects of N2O are similar to those of CO₂. The direct myocardial depression produced is balanced by simultaneous sympathetic stimulation. N2O may increase sympathetic outflow from the brain and inhibit removal of nor-epinephrine by the lung. In intact animals (along with humans) this sympathetic stimulation, coupled with a mild capacity to produce direct depression, results in comparatively little cardiovascular depression at both analgesic and anaesthetic partial pressures. The absence of a marked depression of myocardial contractility and blood pressure distinguishes N2O from the potent inhaled anaesthetics. N2O slightly increases pulmonary vascular resistance and has little effect on hypoxia-induced pulmonary vasoconstriction.

In summary, N2O inactivates liver methionine synthase. Very limited evidence suggests that this or other factors related to N2O anaesthesia may lead to hepatic injury. In terms of effects on the kidney, there is little evidence to suggest that N2O may be nephrotoxic. In terms of haematological effects, N2O can produce abnormalities in bone marrow activity as a result of interference with enzymes containing vit B12, which are necessary for the DNA synthesis required to form red blood cells. Whilst not examined in the repeat dose toxicity studies, prolonged exposure to N2O can potentially result in megaloblastic haematopoiesis and leukopenia resulting from the principal inhibition of methionine synthase. Inhibition of this enzyme would appear to be temporary, reversing when exposure is withdrawn.

References:

Eger II, E.I. (1985). Nitrous oxide / N₂O. Elsevier Science Publishing Co., Inc. New York, USA. ISBN 0-7131-4461-0

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
As N2O is a gas, the oral route of exposure is not relevant

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
From the available published data this study, whilst limited in its study design is deemed to be the best study to provide details regarding the repeat dose toxicity of N2O. As the DNELs for N2O are based on occupational health limits derived primarily from human data a specific repeat dose toxicity study in animals would be of limited value and is not considered to be justified.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
As dinitrogen oxide is a gas, the dermal route of exposure is not relevant.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
As dinitrogen oxide is a gas, the dermal route of exposure is not relevant

Insufficient for classification as the toxic effects were observedat extremely high doses which are considered irrelevant for human exposure.

STOT SE H336: May cause drowsiness or dizziness based on the anaestheic properties.