[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rabbit

Quality of whole database:

1

Additional information

In teratology studies in rats and rabbits performed on an analogue family member, the Tetrakis(hydroxy)methyl phosphonium (THPS), maternal toxicity was observed only at the highest tested doses and was characterised by clinical signs, variation of bodyweight gain and necropsy findings similar to those observed during the repeated dose studies, such as hair loss, fur staining, decrease of bodyweight or bodyweight loss, coloured/discoloured organs. In addition, food consumption was markedly affected in animals treated at the high dose (mainly in rabbit species).

 

Solvay hypothesis

This maternal toxicity in the high dose group was not associated with effects on the pre/post implantation rate, number of living foetuses, on the sex ratio, or, on the foetal weight. Foetal malformations were observed at the highest tested dose for rabbits: eye defects in 35% and limb abnormalities in 15% of the living pups. An eye malformation (only 1 foetus with anophthalmia) was observed in rats; as the frequency of this observation in treated animals is comparable to historical control data in this strain and always at a dose level eliciting marked maternal toxicity, it can be concluded that this observation is not caused by THPS.

Moreover, at the same dose levels and in both species, the number of extra thoraco-lumbar ribs (defined as a developmental variation) was significantly increased. This variation was not considered to adversely affect survival or health of the offspring. In addition, these findings were significantly increased at a dose which was considered to be toxic in both species. Supernumary ribs findings are described as being of low to moderate concern in the ECETOC monograph n°31. In addition, this finding is considered related to maternal stress, therefore we do not consider it as being a direct effect of THPS on the foetus.

Eye and limb malformations were noted at the high dose level in which marked maternal toxicity was seen in the rabbit. In the mid and low-dose groups, where no maternal toxicity was observed, no developmental toxicity was seen.  There appears to be an association between the high dose individual maternal toxicity in terms of changes in food consumption and incidence of major abnormalities in the foetuses in the corresponding litters. Moreover, maternal toxicity as measured by food consumption and body weight in this study was likely underestimated because repeated dose toxicity studies in rats revealed significant liver toxicity at similar or lower dose levels.

Indeed, the 28-day toxicity rat study showed clearly that a dose level of 60 mg/kg/day enhanced hepatotoxic effects (i.e. 3/10 animals had pale and mottled livers). In addition, animals of the 90-day toxicity rat study showed also liver toxic effects at lower dose levels (i.e. 10 mg/kg/day) than doses selected for the teratology studies. Given the important role of the liver ensuring the maternal homeostasis, the possible influence of the hepatotoxic effect of THPS deserves further investigations. This indicates that the observed developmental effect could be secondary to hepatotoxic effects. In public literature, other substances showing similar eye and limb malformations were identified (cyanazine, 2,4-dichlorophenoxyacetic acid, flurprimodol). These effects were only observed in excess of maternal toxicity and were assessed not to be a proven direct effect of the test substance.

Pharmacokinetic studies (see section 8.8) described that THPO and formaldehyde are the main metabolites of THPS. We may therefore suppose that foetus are exposed to both molecules. Nevertheless, available literature on formaldehyde (Reach dossier disseminated by ECHA) does not show an association with enhanced eye and limb malformations. Limited data is available on THPO, but available data suggest that THPO is a molecule with a low reactivity and when conducting an analysis based on structural similarity, no alerts for developmental toxicity were observed (QSAR tool box 3.2).

THPS is corrosive to the eye and causes severe dermal irritation following repeated topical administration in rats. Similar effects probably occurred during the gavage of the rats and rabbits teratology studies. This effect might not always have been detected due to the post treatment period and the rapid renewal capability of the intestinal tract. This irritation might have affected intestinal function and combined with the reduced food intake might have resulted to a low absorption of nutrients critical to the foetus. Indeed,female n° 96 of the rat teratogenicity study which delivered a foetus with anophthalmia showed a marked food consumption decrease and had also a duodenal ulcer/abnormal ingesta noted at necropsy.

 

These observations suggest that maternal toxicity is the main driver of these abnormalities. Therefore, additional experiments were performed to elucidate how secondary non-specific effects are occurring and are summarized in the below paragraph. Indeed Solvay has conducted a research project to consolidate the supporting evidence. In addition, there is a clear toxicological threshold for the developmental effect, below which no effects would be expected. As findings were only observed in the presence of significant maternal toxicity in rabbits, THPS active substance has been self-classified into category.2 H361d. (Suspected of damaging the unborn child)

 

Solvay results

Tetrakis(hydroxymethyl)phosphonium sulphate (THPS) induces birth defects (mainly eye and skeleton malformations) in rabbits. These malformations showed similarities to those caused by vitamin A deficiency. In addition, in vivo studies in other animal species and ex vivo studies in rat and rabbit liver slices revealed liver toxicity caused by THPS. In the present study we hypothesized that deregulation of vitamin A homeostasis in the liver in combination with decreased food intake/uptake by rabbit dams leads to decreased supply of vitamin A to the developing foetus, explaining the birth defects. For this purpose New Zealand White (NZW) rabbits were exposed to THPS 75% at 0, 18 or 60 mg/kg/day during day 7 -19 after coitus for evaluation of liver and intestinal toxicity and of its influence on vitamin A homeostasis.

Rabbit dams of the highest dose group had a significantly reduced intake of food and water from the start of the study (from day 7) until the day of necropsy, whereas in the 18 mg/kg group food intake was only low between gestation day 12 -18. In addition, THPS at 60 mg/kg induced adverse effects on the liver (triglyceride accumulation and periportal cytoplasmatic inclusions) and a significant increase of serum Gamma Glutamyl Transferase (GGT). No indications were found for acute liver necrosis or inflammation. The gastro-intestinal tract seemed to be unaffected. The retinol content of the liver of the dams was dose-dependently (although not statistically significantly) decreased. Liver retinyl palmitate levels on average were unchanged. In addition, the liver content of essential transporter proteins for retinoids, retinol binding proteins (RBP)1 and RBP4 was significantly decreased. as was the gene expression of lecithin retinol acyltransferase (LRAT, that catalyses production of retinyl esters for storage) and cytochrome p450 (cyp)26A1 (that mediates catabolism of the biologically active all trans retinoic acid). All these observations suggest a physiological response to retain vitamin A homeostasis under conditions of (apparent) vitamin A deficiency. Also, serum retinol content as well as the serum concentration of the transporter protein RBP4 were significantly decreased by 25-50% (at the end of study) in both treated groups relative to the control. Since the developing foetus in the stage of organogenesis solely depends on the mother ́s supply of retinol (in the fasting state) and retinyl palmitate (in case of sufficient food intake), this could have resulted in vitamin A deficiency in the foetuses, particularly in the highest dose groups. Preliminary measurements were done to relate the effects in the mothers to those in the pups at the day of necropsy. Retinoids in the liver of the foetuses at the end of study showed significant and dose-dependent reduction of retinol and retinoic acid content in the highest dose group, whereas retinyl palmitate storages overall were unchanged. In addition, a significant increase of (retin)aldehydrogenase activity was observed in the liver.

In conclusion, THPS exposure affected food intake of the dams and caused adverse effects on the liver. In addition, vitamin A homeostasis was dysregulated in both mothers and pups.

Together these observations could explain the birth defects in earlier teratogenicity studies.THPS acts indirectly and is therefore considered not to be responsible of the teratogen events described in the rabbit species.

 

This Solvay's work has been accepted for publication in the Journal of Applied Toxicology. The title of the manuscript is "Disruption of vitamin A homeostasis by the biocide Tetrakis(hydroxymethyl) phosphonium sulphate in pregnant rabbits".

Justification for selection of Effect on developmental toxicity: via oral route:
One reliable data is available for developmental toxicity. There is one study performed in rabbit (equivalent to OECD 414 test method, Kr.1). This study was chosen as the key study for this endpoint.

There is also an mechanistic study available to explain the mechanism of action of the reprotoxicity observed in the OECD 414 rabbit study

Justification for classification or non-classification

The study performed on an analogue of the substance (THPC-Urea) revealed that adverse reprotoxicity findings were observed only at maternal dose toxicity. Mechanistic studies performed on another analogue of the substance, i.e THPS, revealed that THPS acts indirectly for the reprotoxicity which confirmed that THPS-urea monomer should be classified into category 2, H361d (Suspected of damaging the unborn child) according to CLP (1272/2008) criteria.