Dinitrogen oxide

Endpoint summary

Description of key information

Nitrous oxide is not expected to induce adverse effects in the haematopoietic system at occupational concentrations and under occupational exposure scenarios.

Since nitrous oxide is a gas, the oral and dermal route is not relevant for the assessment of repeated dose toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Limited haematology and pathology

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

4 h/d, 5 d/w

Dose / conc.:

5 000 ppm

Remarks:

(0.5%)

Dose / conc.:

50 000 ppm

Remarks:

(5%)

Dose / conc.:

500 000 ppm

Remarks:

(50%)

No. of animals per sex per dose:

27

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly
HAEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Liver microsomal cytochrome P-450 content and the rates of defluorination of enflurane and methoxyflurane were determined.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment related decreases in body weight were observed at 500 000 ppm 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).

Haematological findings:

no effects observed

Description (incidence and severity):

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

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

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

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment related changes were observed in any of the parameter measured. No impairment of the testis weight was observed.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histological changes in the liver, kidneys or adrenal gland were detected.

Other effects:

no effects observed

Description (incidence and severity):

Neither the hepatic microsomal cytochrome P-450 content nor the rates of anesthetic defluorination were increased. No enzyme induction or inhibition could be observed.

Key result

Dose descriptor:

NOAEC

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponds to 90909 mg/m3

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

90 909 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

20

Species:

mouse

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose: oral and dermal route

Since nitrous oxide is a gas, the oral and dermal route is not relevant for the assessment of repeated dose toxicity.

Repeated dose: via inhalation

Animal studies reflecting exposure duration and concentrations relevant for the workplace are not available. Occupational exposure to nitrous oxide without waste gas scavenging usually ranges between 400 and 3000 ppm (ACGIH report, 2001). Whilst using concentrations of 5000 ppm and more, the study of Rice et al. (1985) is considered the only relevant investigation for the assessment of systemic effects after long-term exposure to nitrous oxide.

Swiss Webster mice (15/sex/gp) were exposed to nitrous oxide via whole body inhalation at concentrations of 0, 5000, 50 000 or 500 000 ppm [0, 0.5, 5, 50%] for 4h/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 nitrous oxide, or more likely that continuous exposure is necessary to induce leucocytopenia, as previous demonstrated following continuous exposure to nitrous oxide 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 nitrous oxide, 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%]) (Rice et al, 1985).

Effects on the haematopoietic system observed in man and laboratory animals (MAK value documentation, 1993) occur only at high concentrations usually used for anaesthesia and are therefore not relevant for the hazard assessment at the workplace. They were included in the dossier for the sake of completeness.

After exposure of rats to 10 000 ppm nitrous oxide for 5 weeks (6 hours/day, 5 days/week), the number of mast cells in the peripheral blood and bone marrow was increased. After 9 weeks normal values were found once more. Exposure for 6 months resulted initially in a reversible polycythaemia and, after 5 months, in an increase in the number of reticulocytes. The meaning of these results is not clear. No leukocytopenia was observed (Cleaton-Jones et al. 1975, 1977).

After up to two weeks continuous exposure to 500 000 ppm there was a significant reduction in the leukocyte and lymphocyte counts, in the number of nucleated mature bone marrow cells, pluripotent stem cells and granulocyte precursors in the bone marrow of splenectomized mice (Suzuki et al. 1990).

In rats exposed intermittently (21 to 35 days, no other information) to nitrous oxide concentrations of 200 000 ppm the only effect observed was a decrease in the lymphocyte count. With continuous administration, the leukocyte, reticulocyte and lymphocyte counts were reduced after 35 days. After day 10, changes in the bone marrow occurred. The same effects in the bone marrow were seen after as little as three days intermittent or continuous exposure to 400000 ppm (Kripke et al. 1977).

Continuous exposure of rats to nitrous oxide concentrations of 800 000 ppm for 2, 4 and 6 days led to a decrease in the white blood cells counts and to changes in the histology of the bone marrow (Green and Eastwood 1963). No NOAEC was described.

Taken together, nitrous oxide is not expected to induce adverse effects in the haematopoietic system at occupational concentrations and under occupational exposure scenarios.

General remark

1 mL/m³ (ppm) = 1.83 mg/m³

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The criteria for classification for specific target organ toxicity after repeated exposure according to Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/1182, are not met.