Registration Dossier

Administrative data

Description of key information

The toxicity after repeated exposure was addressed by data on a substance analogue, Amphoacetates C8 -C18. Two 28 days repeated dose studies were performed, with the mono-acetate and the di-acetate form (containing both mono-acetates and diacetates). The rationale to perform the test with both forms was to demonstrate that both substances are of low toxicity and to demonstrate that the toxicological hazard of both forms are covered in the registration. The outcome of the study is read across to Amphoacetates C8 (see Section 13 for detailed explanation).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Animals of the 1000 mg/kg bw/day group and, on only a few occasions, in animals of the 300 mg/kg bw/day group: Observed salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Rales were noted in several males and females of the 1000 mg/kg bw/day group (and in a single male and female of the 100 mg/kg bw/day group). As these animals showed rales on only one or a few days, this finding was considered not to be toxicologically relevant (it was likely related to the dosing technique).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male and two females died in the high dose group. Mortality was considered to be associated to the dosing technique and not test-item related.
The male was euthanized in extremis on day 17 of the treatment period (day 3 of mating period). Body weight gain was normal up to day 15. Necropsy showed macroscopic findings such as mucous contents in the trachea and gas distended parts of the gastrointestinal tract. Microscopic findings showed acute inflammation of the trachea.
One female was found dead on day 16 of the treatment (day 1 of the post-coitum period). The weight gain during the whole period was poor. Macroscopic findings were swollen lungs; microscopic findings were alveolar content and congestion of the lungs, marked bronchial mucosal erosion and erosion/ulceration of the trachea.
One female was euthanized in extremis on day 40 of the treatment (day 1 of lactation period). Food consumption and weight gain was reduced since day 17-20 of the gestation period. Macroscopic findings were pale liver and greenish kidneys. Microscopic findings were marked ulceration in the forestomach and lymphogranulocytic inflammation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and body weight gain were considered not to be adversely affected by treatment. A few findings at 1000 mg/kg bw/day were regarded as not toxicologically relevant as explained below.
Two males in the dose groups 1000 mg/kg bw/day showed considerably reduced body weight gain over the 4-week treatment period whereas the other males of this dose group grew normally. There were no associated signs of toxicity and mean body weights of 1000 mg/kg bw/day males remained close to control values (4% difference at the end of the treatment period, not statistically significant).
Findings of note in 1000 mg/kg bw/day females consisted of slightly lower mean body weight gain in the last week of the gestation period and reduced weight gain or slight weight loss in three females during the lactation period. Mean body weights of 1000 mg/kg bw/day females did not differ statistically significantly from those of controls (5% difference at the end of the post-coitum and lactation periods). Except for the female which was euthanized in extremis on Day 1 of the lactation period), the lower weight gain was not associated with signs of toxicity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females in the high dose group showed reduced food consumption (before and after correction for body weight) in two periods: Periods between days 14-20 of the post-coitum period (about 10%, statistically significant) and during the lactation period (statistically significant between Days 7-13; mean absolute food consumption in this interval was 20% lower than the control value).These findings were considered not to be toxicologically relevant as they were not associated with an adverse effect on body weight gain.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related changes in red blood cell parameters, white blood cell parameters or number of platelets.
Males treated at 1000 mg/kg bw/day had slightly lower activated partial thromboplastin time (APTT) values than concurrent controls. As all values at 1000 mg/kg bw/day remained within the historical control range, this change was regarded as non-adverse.
Female rats showed no treatment-related changes in coagulation parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry parameters showed no differences between control and treated rats that were considered to be toxicologically significant.
Mean values for alanine aminotransferase (ALAT) and bile acids in males treated at 1000 mg/kg bw/day were higher compared to the control group means (40 and 77%, respectively). These differences were not statistically significant, mean values at dose group 1000 mg/kg bw/day remained in the historical control ranges, and there were no associated anatomic pathology alterations. As such, these clinical chemistry findings were regarded as non-adverse.
Isolated, statistically significant variations noted in clinical chemistry parameters (higher creatinine and sodium in males at 300 mg/kg bw/day) were considered to be unrelated to treatment due to the lack of a dose-related trend and/or small magnitude of the difference from controls.
Serum levels of T4 in males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was not affected by treatment. A statistically significantly higher forelimb grip strength noted in males at 300 mg/kg bw/day was judged to be unrelated to treatment due to the lack of a dose-related trend.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. In the absence of a dose-related trend, the higher values for total movements and ambulations noted in 100 mg/kg bw/day females, particularly two of them, were regarded as unrelated to treatment.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In females at 1000 mg/kg, a statistically significantly lower mean absolute brain weight (relative difference 7%) was regarded as unrelated to treatment. Brain weights of 1000 mg/kg females were close to the historical control mean (except for one female whose brain weight was at the lower end of the historical range), whereas most concurrent control values were at the higher end of the historical range. Moreover, the brain to body weight ratios did not differ statistically significantly. For these reasons, the intergroup difference in absolute brain weight was considered to reflect normal background variation rather than an effect of the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain, and were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.
Key result
Critical effects observed:
not specified

Analysis of dose preparations: The concentrations analyzed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 93% and 100%). A small response noted at the retention time of the test item in the chromatograms of the control group formulation was considered to derive from carry-over in the analytical system. This had no significant effect on the results of the other study samples because of the minor magnitude (maximally 0.0092% relative to low dose group samples). The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation ≤5.8%).

Summary results dose range finding study: Mortality was not observed. At 500 and 1000 mg/kg bw/day, slight salivation was noted after dosing. At 250 mg/kg bw/day, reduced weight gain in one animal was noted. At 500 mg/kg bw/day, weight loss was found in one animal between Days 5-11, in the other two reduced weight gain was found. At 1000 mg/kg bw/day, weight loss was seen in one animal, the other two were normal. No effects were seen on food consumption and at macroscopic examination. Kidney weights were normal in all rats, liver weights were slightly higher in one animal in each group at 250 and 1000 mg/kg bw/day each.

Conclusions:
An oral Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening was performed according to OECD/EC guidelines and GLP principles with substance analogue Miranol Ultra C32 (Amphoacetates C8-C18). No adverse effects were observed and therefore the NOAEL was established to be 1000 mg/kg bw/day. This result is read across to Amphoacetates C8.
Executive summary:

A combined oral repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Substance analogue Amphoacetates C8 -C18 (Miranol Ultra C32) was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were treated for 29 days, up to and including the day of the scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 or 15 days after delivery, up to and including the day before scheduled necropsy. Females without offspring were treated for 53 days (no evidence of mating) or 42-43 days (not pregnant or implantation site only). Treatment with Miranol Ultra C32 was associated with a few non-adverse changes at the highest dose group i.e. slight salivation in both sexes, lower food consumption in females in the last week of gestation and during lactation, and lower activated partial thromboplastin time in males. No treatment-related or toxicologically relevant changes were noted in the other parameters investigated in this study. Based on the absence of adverse effects up to 1000 mg/kg bw/day, a parental No Observed Adverse Effect Level (NOAEL) for Miranol Ultra C32 of 1000 mg/kg bw/day was established. This result is read across to Amphoacetates C8.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study has Klimisch score 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

A combined oral repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Substance analogue Amphoacetates C8 -C18 (Miranol Ultra C32, mono-acetate form) was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were treated for 29 days, up to and including the day of the scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 or 15 days after delivery, up to and including the day before scheduled necropsy. Females without offspring were treated for 53 days (no evidence of mating) or 42-43 days (not pregnant or implantation site only). Treatment with Miranol Ultra C32 was associated with a few non-adverse changes at the highest dose group i.e. slight salivation in both sexes, lower food consumption in females in the last week of gestation and during lactation, and lower activated partial thromboplastin time in males. No treatment-related or toxicologically relevant changes were noted in the other parameters investigated in this study. Based on the absence of adverse effects up to 1000 mg/kg bw/day, a parental No Observed Adverse Effect Level (NOAEL) for Miranol Ultra C32 of 1000 mg/kg bw/day was established.

A second oral Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening was performed according to OECD/EC guidelines and GLP principles with the diacetate from of substance analogue Amphoacetates C8 -C18. Wistar Han rats were treated with Dehyton®DC (diacetate form) by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 50-54 days). Females that failed to deliver pups were treated for 41 days, with the exception of females in the 1000 mg/kg bw/day dose group that were euthanized or found dead (one female) at the end of the premating period or during the post-coitum period due to adverse clinical observations. Accuracy and homogeneity of formulations determined by chemical analyses confirmed accurate dosing. At 1000 mg/kg bw/day, there was a high mortality in the females (4/10) and one premature death in the males. These deaths were concluded to be related to regurgitation and thus secondary to the test item (possibly triggered by physical/chemical properties of the test-item solution in combination with the route of administration). Follicular cell hypertrophy of the thyroid gland was found in males at the 1000 mg/kg bw/day dose group. Similar findings were observed at the 300 mg/kg bw/day dose group but at a slightly lower severity. These findings were considered to be non-adverse based in its low severity (up to mild) and absence of any additional degenerative, inflammatory or proliferative findings and changes in T4 hormone levels. Based on these results, the no-observed-adverse-effect level (NOAEL) was found to be 300 mg/kg bw/day.

It is of note that a sub-chronic toxicity study was performed according to OECD/EC guidelines and GLP principles with Dehyton, based on which it was concluded that the the no-observed-adverse-effect level (NOAEL) for sub-chronic exposure was 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data, Amphoacetates C8 is not classified for toxicity after repeated exposure according to CLP Regulation (EC) No. 1272/2008.