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Administrative data

Description of key information

1 year chronic, oral, dog study, 90 day chronic, oral, dog study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
11 mg/kg bw/day
Study duration:
chronic
Species:
rat

Additional information

The repeated dose oral toxicity of AMP has been assessed in multiple species (Monkey, dog, rat, mouse), over multiple dosing periods (5 day, 28 day, 90 day, 1 year). In all species, oral dosing was well tolerated, although animals dosed in a study where the pH of the test material was 11 showed evidence of significant gastrointestinal irritation and distress due to the high alkalinity of the test material. With the exception of the 8 -week study in mice (where no significant toxicity was observed), the liver appears to be the target organ for AMP. Specifically, AMP appears to cause a dose related accumulation of lipids in hepatocytes, and this is linked to an increase in liver weight and presence of liver enzymes in the blood in the more severely affected animals. There appears to be an increased sensitivity to this effect in females compared to males. There is also a difference in species sensitivity, dogs = rats > mice.

With the exception of liver toxicity in rats and dogs, no other significant toxic effects were noted (excluding local effects caused by the alkalinity of the test material) in the repeated dose studies.

The toxicity of AMP observed in the repeated dose Dog studies is consistent and as indicated above, involves the liver as a primary target organ. In the 28-day study, the lowest dose tested (19 mg/kg bw) appeared to be a no observed adverse effect level due to liver toxicity observed in the mid and high dose groups, however even at this dose there was some evidence of a minor (not adverse) liver effect in the female dog (the male dog showed no signs of effects).

In the 3 month study similar effects as those observed in the 28-day study were observed, however whilst there were minimal effects in the dogs dosed with approximately 15 mg/kg bw/day, one female dog dosed with 0.63 mg/kg bw had signs of liver toxicity consistent with those seen in the highest dose group (approx 63 mg/kg bw). This evidence of toxicity was considered to be either a spurious finding, evidence of a hyper-sensitive animal or errors in the dosing of the animals. Due to the low number of animals used in this dog study (4 per sex per dose) it was difficult to conclude whether this effect should be considered to be treatment related.

In the 1-year dog study, the highest dose tested (2.8 mg/kg) was the no effect level. The lack of any treatment related toxicity in this study suggests that the effects observed at 0.63 mg/kg bw in the 3 month study were more likely to be spurious than a real treatment related effect. It is however unfortunate that the dose used was so low and did not allow an assessment of whether a dose closer to 15 mg/kg bw/day was the NOAEL.

A 90-day rat study was conducted in tandem to the 90-day dog study and the NOEL from that study was approximately 15 mg/kg bw/day, with liver toxicity being the primary toxic effect observed at the higher doses. This consistency between the two species in NOAELs indicates that they are equally sensitive, or that the Dog (a larger animal) is perhaps slightly less sensitive to the liver toxicity, since the allometric differences should result in a lower NOEL in Dogs compared with Rats.

Given the available toxicokinetic information on AMP in the rat, and the consistency between the rat and the dog in NOELs from comparable studies it is proposed to use the NOAEL from the 2-generation study performed using the associated chemical 4,4-dimethyl oxazolidine. This substance hydrolyses almost immediately in the stomach following oral dosing; releasing formaldehyde and AMP (refer to summary of reproductive toxicity). In this study the rat the NOEL, the equivalent dose of AMP administered was 11 mg/kg bw/day. Again, the primary systemic toxic effect observed was hepatotoxicity at the next highest dose. The preference for using this study, and not the repeated dose rat or dog studies, is that the available toxicokinetic information allows a far more accurate extrapolation from the oral dose route used in the study to the dermal exposure route in humans when deriving a DNEL. With respect to extrapolating from the 2 -generation study to a long term human exposure situation, a factor of 1 is considered sufficient due to the length of this study, and the consistency of the results with the sub-chronic and chronic rat and dog studies.

Probable Mode of Action

Choline and phosphatidyl choline are key in the transport of triglycerides from the hepatocytes in the form of VLDL (very low density lipoprotien). Interfering with the manufacture of phosphatidyl choline thus limits the capacity of the liver to transport lipids. The effect of AMP on the liver appears to be a result of interference with phospholipid synthesis in the hepatocytes. Various publications on AMP from the 1950's and 1960's have identified that it is capable of becoming incorporated into phospholipids in place of ethanolamine and/or choline and that it inhibits the uptake of choline by the liver cells. AMP also appears to inhibit the formation of choline in the liver via the conversion of ethanolamine to choline. It is possible that a phosphatidyl AMP moiety is competing for the enzyme phosphatidyl ethanolamine methyl transferase (converts phosphatidyl ethanolamine to phosphatidyl choline), preventing the formation of phosphatidyl choline, however it is as yet unclear as to the exact mechanism. What is however clear is that in the presence of suficient dietary choline, the effects of AMP on the liver are prevented, and this is likely due to the lower dependance on de novo choline synthesis when sufficient choline is present in the diet. Thus the hepatotoxicity is in part dependent on the presence of sufficient choline in the diet.

The transport of lipids from hepatocytes is fairly consistent through out mammalian physiology, i.e. the use of a phospholipid based transport such as VLDL. Therefore the effects observed in rats and dogs are likely relevant to man. However the increase in hepatic lipids is only the first step in the toxicity to the liver, and further indicators of toxicity are only evident after some accumulation of lipids. There is no evidence of liver toxicity (e.g. increased liver weight, increase in liver enzymes in the blood etc.) at doses where lipid accumulation in hepatocytes is not apparent . It is also apparent that the liver toxicity is the most sensitive endpoint, occurring at doses lower than those causing other effects, such as increased post implantation loss (refer to reproductive toxicity section).


Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Classification for Chronic toxicity or Target Organ toxicity is proposed. STOT RE Cat. 2 (Liver) is proposed based on histopathological effects on Liver in multiple repeated dose toxicity studies.