Registration Dossier

Reference

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, described as a dominant lethal test. Testicular effects investigated

Qualifier:

no guideline available

Principles of method if other than guideline:

Described as a dominant lethal test. Testicular effects investigated

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on mating procedure:

At the end of the period the males were each housed with untreated 2 females for 5 days. All females were killed 14 d later and the uterine contents examined.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

9 wks

Frequency of treatment:

6 h/d 5 d/wk for 9 wk (i.e. over a whole spermatogenic cycle)

Details on study schedule:

Groups of 15 M SD rats were exposed to 0, 0.1, 0.5 or 1.0% N2O in air for 6 h/d 5 d/wk for 9 wk (i.e. over a whole spermatogenic cycle). At the end of the period the males were each housed with untreated 2 females for 5 days. All females were killed 14 d later and the uterine contents examined.

Remarks:

Doses / Concentrations:
0, 0.1, 0.5, 1.0%
Basis:
nominal conc.

No. of animals per sex per dose:

up to 15M/gp

Control animals:

yes, concurrent no treatment

Dose descriptor:

NOAEC

Effect level:

ca. 1 000 ppm

Sex:

male

Reproductive effects observed:

not specified

Executive summary:

Described as a dominant lethal study, groups of 15 M SD rats were exposed to 0, 0.1, 0.5 or 1.0% N2O in air for 6 h/d 5 d/wk for 9 wk (i.e. over a whole spermatogenic cycle). At the end of the period the males were each housed with untreated 2 females for 5 days. All females were killed 14 d later and the uterine contents examined.

A dose related trend towards increased resorption rate with increasing dose of N2O and a slight dose-related reduction in the number of live fetuses. whilst the differences were not statistically significant, the NOAEC for fertility effects on the testes can be considered to be 1000 ppm, the lowest dose tested in all the fertility effects.

Endpoint conclusion:

no study available

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 830 mg/m³

Study duration:

subacute

Species:

rat

Endpoint conclusion:

no study available

No suitable studies on N2O have been identified to address the endpoint of reproductive toxicity (fertility and sexual function) under the requirements of REACh. The clinical relevance of the published studies remains doubtful because of the extremely long duration of exposure and high doses used.

N2O is known to oxidise vitamin B12 rendering it inactive as a coenzyme in many essential metabolic processes. One vitamin B12 dependent enzyme in particular, methionine synthase is essential for normal cell division. Prolonged exposure to N2O leading to inactivation and dysfunction of methionine synthase in the male or female germ cell could then have adverse effects on fertility of on the growth of the developing fetus. Thus a mechanism exists to explain the association between exposure to N2O and both male and female reproductive toxicity.

Histological changes, typified by depletion of spermatogenic cells have been identified in the testes of rats following continuous exposure to 20% N2O for up to 35 d (Kripleet al., 1976). Similar effects however were not observed in mice exposed for 4 h/d for 14 wks to levels of 50% N2O (Mazze et al., 1983). In addition, following a 5 d exposure to 80% N2O for 4 h/d did not cause any morphological changes in mice (Land et al., 1981). The Mazze et al (1983) and Land et al (1993) data would appear to be in contrast to effects reported by various groups who used rats rather than mice as the model to investigate potential fertility effects. Rats exposed to 0.5% N2O for 6 h/d for 30 d showed some evidence of reduced fertility (Vieira et al., 1983).

The data may suggest that the murine model is an insensitive model for investigating potential fertility effects, whilst a mixture of results have been reported in the rat model.

Webman (1980) showed no effect on fertility of either sex of rat when mated with unexposed animals or when exposed animals of both sexes were mated following exposure to 70% N2O for 2 h/d for 30 d. Histological examination of ovaries and testes after this exposure regimen showed no effect. Kugel (1990) found a significant reduction in fertility following only 4 d of exposure to 30% N2O for 8 h/d.

The lowest exposure level tested for fertility effects on the testes has been at 1000 ppm N2O for 6 h/d for 9 weeks (Holson et al.,1995). This may be considered to be a NOEL for these effects in the rat.

From an overview of the available data, it seems that where fertility effects arise from exposure >10% N2O the daily duration of exposure is more important than the total duration of the exposure level. Intermittent exposure up to 8 h/d, 5 d/wk (reminiscent of any potential occupational exposure) is associated with a much lower level of effects than constant exposure to the same N2O concentrations. The effects of N2O on female fertility reported by Kugel et al (1990) on LHRH are unique in that this possible mechanism of action has not previously been investigated nor followed up. One could however point out that as N2O is a commonly used anaesthetic, particularly used during the clinical procedure of assisted fertilisation would provide considerable reassurance regarding potential fertility issues in humans.

As the DNELs for N2O are based on occupational health limits derived primarily from human data a specific reproductive toxicity study in animals would be of limited value and is not considered to be justified.

Justification for selection of Effect on fertility via inhalation route:
The lowest exposure level tested for fertility effects on the testes has been at 1000 ppm N2O for 6 h/d for 9 weeks (Holson et al., 1995). This may be considered to be a NOEL for these effects in the rat.

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

single dose level used

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Gravid rats used

Duration of treatment / exposure:

exposure from GD1 to GD19

Frequency of treatment:

continuous treatment

Duration of test:

19 days

Remarks:

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

No. of animals per sex per dose:

12 gravid rats/gp

Control animals:

yes, concurrent no treatment

Dose descriptor:

LOAEC

Remarks:

Viera (1979)

Effect level:

ca. 5 000 ppm

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEC

Remarks:

Vieira et al (1980)

Effect level:

ca. 500 ppm

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEC

Remarks:

Vieira et al., (1978)

Basis for effect level:

other: developmental toxicity

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Dose descriptor:

NOAEC

Remarks:

Vieira et al (1983)

Effect level:

ca. 1 000 ppm

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Increased foetal loss and resorption was seen in the N2O treated group. Fetal weights and crown-rump length were also decreased in N2O exposed fetuses. Skeletal abnormalities were not seen in the controls but were observed in 9% of foetuses exposed to N2O. These were not described in detail although it is commented that the incidence was greater in females than males. No abnormalities of the internal organs were recorded (Vieira, 1979).

A further study by Vieira et al (1980) continually exposed 12 pregnant Wistar rats from the day after mating until GD 19 to N2O in air at concentrations of 0, 250, 500 and 1000 ppm. At the highest dose tested mean litter size and crown-rump length were significantly reduced compared to the control; other exposure levels showed no difference from the control in these respects. Skeletal abnormalities were only observed in the high dose gp, with soft part abnormalities not reported for any group. The high dose gp contained 4 resorptionsvs. zero in the control gp.

The effects of N2O exposure during different periods of gestation on pup development were investigated. Groups of rats exposed to air containing N2O (10000 ppm) for 6 h/d, 5 d/wk for wk 1 of gestation, the first 2 wks of gestation of for the whole 3 wks of gestation. All rats were allowed to deliver their litters and litters were monitored for 8 wks post partum. The litter size of all the groups exposed to N2O was significantly lower than control and body weight of those pups was lower than that of controls throughout the 8-wks post partum monitoring. Pups from mothers exposed during the 1^stwk of gestation only were light than all others on all occasions. Tail length and body length were reduced for all N2O treated groups throughout the 8 wk post-partum period, compared with controls, although the differences were not always statistically significant (Vieira et al., 1978).

Finally, in a further report by Vieira et al (1983) where gps of pregnant Wistar rats (12/gp) were exposed for 6 h/d, 5 d/wk to 0, 250, 500, 1000 or 5000 ppm in air throughout gestation. Dams were killed on GD 19and the urterine contents were examined. Litter size and mean crown-rump length of the fetus was reduced in the group exposed to 0.5% N2O. There were no malformations or resorptions in any of the groups.

Results from continuous exposure to N2O throughout gestation resulted in significantly more effects than intermittent exposure at similar levels.

Executive summary:

Groups of 12 gravid rats were exposed to a constant level of 0.5% N2) (5000 ppm) from GD 1 through to GD 19. A control group were exposed to air alone (12/gp). Dams were killed on GD 19 and uterine contents examined. Detailed examination of the uterus, ovaries and fetuses were undertaken. Fetuses were fixed, cleared and stained with alizarin red, examined for skeletal anomalies and their crown-rump lengths measured.

Increased foetal loss and resorption was seen in the N2O treated group. Fetal weights and crown-rump length were also decreased in N2O exposed fetuses. Skeletal abnormalities were not seen in the controls but were observed in 9% of foetuses exposed to N2O. These were not described in detail although it is commented that the incidence was greater in females than males. No abnormalities of the internal organs were recorded (Vieira, 1979).

A further study by Vieira et al (1980) continually exposed 12 pregnant Wistar rats from the day after mating until GD 19 to N2O in air at concentrations of 0, 250, 500 and 1000 ppm. At the highest dose tested mean litter size and crown-rump length were significantly reduced compared to the control; other exposure levels showed no difference from the control in these respects. Skeletal abnormalities were only observed in the high dose gp, with soft part abnormalities not reported for any group. The high dose gp contained 4 resorptionsvs. zero in the control gp.

The effects of N2O exposure during different periods of gestation on pup development were investigated. Groups of rats exposed to air containing N2O (10000 ppm) for 6 h/d, 5 d/wk for wk 1 of gestation, the first 2 wks of gestation of for the whole 3 wks of gestation. All rats were allowed to deliver their litters and litters were monitored for 8 wks post partum. The litter size of all the groups exposed to N2O was significantly lower than control and body weight of those pups was lower than that of controls throughout the 8-wks post partum monitoring. Pups from mothers exposed during the 1^stwk of gestation only were light than all others on all occasions. Tail length and body length were reduced for all N2O treated groups throughout the 8 wk post-partum period, compared with controls, although the differences were not always statistically significant (Vieira et al., 1978).

Finally, in a further report by Vieira et al (1983) where gps of pregnant Wistar rats (12/gp) were exposed for 6 h/d, 5 d/wk to 0, 250, 500, 1000 or 5000 ppm in air throughout gestation. Dams were killed on GD 19and the urterine contents were examined. Litter size and mean crown-rump length of the fetus was reduced in the group exposed to 0.5% N2O. There were no malformations or resorptions in any of the groups.

Endpoint conclusion:

no study available

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

900 mg/m³

Study duration:

subacute

Species:

rat

Endpoint conclusion:

no study available

Developmental toxicity associated with N2O exposure has been reported in a wide range of animal studies. Although there are a number of developmental toxicity studies in rodents very few are considered suitable for regulatory submission. There are currently no data which satisfy the requirements for a developmental toxicity study in the rabbit. A review of available published animal data confirms that exposure to atmospheres containing 500 ppm N2O show no evidence of any reproductive effects in animals, even when exposures are continuous throughout pregnancy. Male fertility is also unaffected at similar exposures. Foetal loss has been seen in rats following constant daily exposures to 1000 ppm N2O, but not at 500 ppm. With intermittent exposures (6 h/d, 5 d/wk) such effects are not seen even at 1000 ppm. Since there is strong evidence that methionine synthase is the underlying mechanism of the reproductive effects in animals this lack of effect at 500 ppm would be consistent with the observations that there is no effect on activity of this enzyme at this exposure level, even after chronic exposure.

Even if the NOAEL was identified from a guideline compliant study it is difficult to see how this information would be used as the DNELs for N2O are derived from human data. Therefore, further studies on the developmental toxicity of N2O are not required.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Since there is strong evidence that methionine synthase is the underlying mechanism of the reproductive effects in animals this lack of effect at 500 ppm would be consistent with the observations that there is no effect on activity of this enzyme at this exposure level, even after chronic exposure (Vieria et al., 1980)

No futher data

Whilst animal data shows evidence of adverse fertility and developmental effects, these occur at extremely high doses which following prolonged / continuous exposure. Consequently the exposure scenarios and doses used are not typical of occupational exposure and are therefore deemed insufficient for classification.