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Effects on fertility

Additional information

Toxicity to fertility:

Trioxane has not been tested for effects on fertility in a standard OECD guileline testing protocol. However several studies are available to address the potential effects of trioxane on fertility.

In dominant lethal assays male rats were exposed by gavage and inhalation (Baranski et al., 1984). The assays were performed with an exdended protocol including longer exposure times and histopathology of the testes.

With high oral doses of 850 and 1700 mg/kg bw trioxane for 8 weeks, no influence on reproductive parameters (resorptions, implants, live fetuses etc.) and testes weights was observed. Effects on fertiliy were adressed in a weekly mating design. Although no functional deficit in fertility was recognized in males, histopathology of testes revealed focal necrosis of seminiferous epithelia and testicular lesions. This observation was reported in 1/10 control animals, 3/10 males of the 850 mg/kg bw dose group and 2/10 high dose males. An unequivocal dose dependency can therefore not be established and both dose groups exhibit significant signs of maternal toxicity.

With inhalative exposure of male rats for 12 months at a dose level of 2500 mg/m3, similarly, no treatment related effect on male fertility was described. Furthermore no histopathological changes in the testes were described and the study did not reveal induced dominant lethal mutations in germ cells. Males were mated with females at the end of the exposure, and necropsy of the mated females was performed on day 13/14. No changes were seen in preimplanation losses, dead implants, or the number of live fetuses when compared to control.

  

The effect of trioxane exposure on the oestrus cycle of female rats was assayed following a 7 weeks gavage period with 190, 580 or 1160 mg/kg bw/day of an aqueous trioxane solution (Sitarek & Baranski, 1990). At the high dose level (1160 mg/kg bw/day) a reversible prolongation of oestrus cycle was observed in the 6thand 7thweek of treatment, although accompanied by signs of toxicity, e.g. decrease in body weight, vocalization or apathy. Therefore no effect on the female reproductive cycle was observed unless other overt signs of toxicity occurred, providing a strong indication that trioxane does not interfere with ovarian function. The NOAEL for systemic toxicity in this assay was identified as 580 mg/kg bw, based on behavioral changes. Decreased body weight was observed at the end of the application period in all dose groups, but the adverse nature of this effect is unclear. The value of this systemic NOAEL for a further refinement of the repeated dose toxicity is low, since the study report is lacking important information e.g. reversibility and food consumption.

 

A 90 day oral gavage study with rats was performed according to the current OECD 408 guideline and trioxane dose levels of 30, 100, 300 mg/kg bw/day (RCC, 2002). According to the guideline this study included a detailed macroscopic and microscopic analysis of all reproductive organs and endocrine glands (e.g. ovaries, testes, seminal vesicles). No significant test substance related effects were observed.

In summary the dominant lethal assays (oral, inhal.) with an extended protocol, the ovarian function assay and the oral 90 -day study provide sufficient evidence, that no effects on female and male reproductive function were observed without systemic toxicity. It is well known, that oestrus cycle length is an indicator for more subtle impairments of fertility (e.g. reduced pup numbers). Histopathological findings in the testes are inconsistent and were only described in the dominant lethal test at dose levels with signs of general systemic toxicity but not in the 28- and 90-day studies. In rodents histopathological lesions in the testes have been shown to be a more sensible parameter of toxicity to fertility than male fertility indeces.

Therefore no classification for fertility is indicated, although provided tests are not conform with current guideline test designs for fertility. 

 


Short description of key information:
Results from a 90 day oral gavage study with rats (including a detailed analysis of all reproductive organs and endocrine glands) together with the extended protocols of 2 dominant lethal studies (oral and inhalation exposure) and an ovarian function test provide adequate information that trioxane does not exhibit a potential for toxicity to fertility.

Effects on developmental toxicity

Description of key information
In a OECD 414 guideline study groups pregnant females were gavaged from day 7 to 20 of pregnancy. The NOAEL for developmental toxicity was 315 mg/kg bw/day based on malformations in the presence of significant maternal toxicity. 
Additional information

Developmental toxicity:

In a OECD 414 guideline study groups of pregnant female rats were gavaged from day 7 to 20 of pregnancy (Hoechst, 1998). Trioxane was applied as a 20% aquaeous solution in dose levels of 100, 315 and 1000 mg/kg bw/day.

Significant maternal toxicity was observed. Decreased corrected bw gain at all dose levels (no NOEL), and decreased absolute bw gain and food consumption in the high dose group were reported. 

The NOEL for developmental toxicity was determined to be 100 mg/kg bw/day based on variations (retarded ossification, increased incidence of wavy or thickened ribs). The NOAEL for developmental toxicity was 315 mg/kg bw/day based on malformations, which were only observed in the high dose group (aplasia of tail, sacral vertebral arch and sacral vertebral centres). In this dose group additionaly body weights and crown-rump length of fetuses were decreased.

Adverse developmental effects were only observed in the range of clear maternal toxicity (1000 mg/kg bw/day).

 

Two additional developmental toxicity studies were published from the polish coworkers. Both experiments were not performed according to the OECD guideline or GLP.

In one experiment developmental effects were assayed. 22-24 pregnant rats were orally dosed with 770, 1550 and 3870 mg/kg bw trioxane from days 8-20 of gestation (Sitarek, et al., 1988). The same report refers to a second experiment in which 190 mg/kg bw trioxane was administered.

Embryonic and fetal lethality, malformations and retarded fetal development were reported in the 1550 and 3870 mg/kg bw dose groups. In parallel severe maternal toxicity was observed indicated by reduced body weight gain and food consumption, histopathological liver changes and alterations in organ weights.

With 770 mg/kg bw an increase of fetuses with hydrocephalus, retarded ossification of the sternum and incompletely developed cranial bones were described. Only the later finding was statistically significant and again in parallel to a 12% decrease in matenal body weight. No effects were observed in a second experiment with 190 mg/kg bw trioxane.

The validity of the study is considerably restricted due to experimental deficiencies, limited reporting and an unexplained increased incidence of morphological chances in the placenta of all animals. Even in the lowest dose tested (190 mg/kg bw) a significantly increased incidence of histopathological changes (21%/7%) or focal necrosis (6%/0%) in the placentas were observed, if compared to control groups.

 

In the second experiment postnatal development was assayed. 190, 580 and 1160 mg/kg bw trioxane were orally administered to pregnant rats every second day from day 2-20 of gestation (Sitarek, et al., 1990). The Maternal toxicity (reduced bw gain, behavioral changes) occured from 580 mg/kg bw, and at 1160 mg/kg bw the litter size was significantly reduced and most newborn died within few days. No externaly visible malformations were observed. Reversible behavioral changes of the pups were only observed in doses which were toxic for the dams and no further indications for a disturbance of postnatal development was found.

 

According to the Dangerous Substances Directive (67/548/EEC) classification as a developmental toxicant is only indicated in case developmental effects were observed without significant maternal toxicity. This observation should result from a valid animal study. Category 3 is indicated when methodological deficiencies limit the value of the bioassay and an evaluation of effects can only be made with restrictions. No classification as a developmental toxicant (or Cat. 3 at the most) is indicated in case solely minor changes of incedences of spontaneous defects, skeletal variations or minor influence on postnatal development of offspring were observed.

Trioxane was legally classified in Annex 1 to Directive 67/548/EEC as Repr. Cat. 3; R63 (Possible risk of harm to the unborn child). Based on the legal basis described above, this classification resembles a borderline decision, since in a reliable developmental toxicity study:

1)     1000 mg/kg bw/day in the range of clear maternal toxicity and

2)     at lower dose levels with less maternal toxicity only variations (e.g. retarded ossification, increased incidence of wavy or thickened ribs) were observed.

 

Justification for classification or non-classification

No classification for fertility is indicated (although provided tests are not conform with current guideline test designs for fertility).

Trioxane was legally classified in Annex 1 to Directive 67/548/EEC as Repr. Cat. 3; R63 (Possible risk of harm to the unborn child). Although this was unequivocally a borderline decision as described in the summary report of the meeting of the commission working group on the classification and labelling of dangerous substances (ECB Ispra, 16 – 18 January 2002,

ECBI/15/02 Rev. 3).