Registration Dossier

Administrative data

Description of key information

The data for repeated dose toxicity are read across from a substance analogue. Two sub-acute repeated dose studies with screening for reproduction and developmental effects were performed according to OECD 422 with two representatives of one analogue, Amphoacetates C8-C18. One study was done with a mono-acetate form, the second study was done with a diacetate Amphoacetate C8-C18. 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. An oral sub-chronic toxicity study was performed according to OECD/EC guidelines and GLP principles. The rationale to read across the data is attached in Section 13.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose:
read-across source
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100, 300 and 1000 mg/kg bw/day incidental abnormal breathing sounds, labored breathing, shallow breathing, increased and decreased respiratory rate were observed in one or two animals per group on several days between Days 35 and 90. Moreover, fur loss, hunched posture and/or erected fur were noted in one or two animals per group on several days between Days 35 and 90. As the observations occurred in one or two rats per dose group and/or the observations were transient and did not persist, the effects were not considered adverse. Salivation was seen among all animals at 300 and 1000 mg/kg bw/day between Days 3 and 92 and incidental ploughing was observed in all males and one female at 1000 mg/kg bw/day directly after dosing. These findings were not considered to be toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). These signs were considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain in females at 100 and 300 mg/kg bw/day and females up to 1000 mg/kg bw/day remained in the same range as controls over the study period. A lower body weight compared to control was observed for males at 1000 mg/kg bw/day from the second study week onwards (0.88x compared to controls, respectively, at end of treatment). A minimal lower body weight compared to control was observed in males at 100 and 300 mg/kg bw/day starting in the second study week onwards (0.93x and 0.95x compared to controls, respectively, at end of treatment).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in females of all test item-treated groups was similar to the control level over the study period. A slightly lower food consumption was seen in males at 100, 300 and 1000 mg/kg bw/day starting in the second week.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters in all test item treated males and females up to 1000 mg/kg bw/day. At 300 and 1000 mg/kg bw/day platelet numbers were increased in males, however these changes occurred within the range considered normal for rats of this age and strain and were therefore considered to be of no toxicological significance. In males, white blood cell count decreased dose-dependently (7.3, 6.5, 6.4 and 6.0 *10E9/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). This was related to a decrease in lymphocytes (5.8, 5.0, 4.7 and 4.5*10E9/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). In absence of pathological effects related to this observation, these effects were not considered adverse. No such effect was seen in females.

Coagulation parameters of treated rats were considered not to have been affected by treatment. The statistically significant changes in prothrombin time in males at 100 and 300 mg/kg bw/day were considered to be unrelated to the test item as these occurred in the absence of a dose-related trend (18.8, 17.1, 17.6 and 17.6 s for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). No such effects were seen in females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical chemistry parameters in all test item-treated females. At 1000 mg/kg bw/day, alkaline phosphatase activity was slightly increased in males, however these changes occurred within the range considered normal for rats of this age and strain and were therefore considered to be of no toxicological significance (115, 123, 138 and 166 U/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). Moreover, cholesterol (HDL and LDL) levels were increased in males, however this slight increase was mainly caused by a single animal and was therefore not toxicologically relevant (1.05, 1.09, 1.13 and 1.22 mmol HDL/L, 0.31, 0.31, 0.31 and 0.45 mmol LDL/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively).
Lower TSH values in all test item-treated male groups (0.376, 0.075, 0.113 and 0.038 uIU/mL for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively) and lower T4 values (4.97, and 4.01 ug/dL for controls and males treated at 1000 mg/kg bw/day, respectively) were observed achieving a level of statistical significance when compared to controls. In the absence of a treatment-related distribution, these values were considered to be of no toxicological significance.
While few other changes in males and females were statistically significant, the alterations in clinical biochemistry parameters were unrelated to administration of the test item due to the minimal magnitude of the change and the absence of a dose response.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights. Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of an effect on final body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations. 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. Stage dependent qualitative evaluation of spermatogenesis in the testes was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 71 up to and including Day 91). One control female, and one female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day showed an irregular estrous cycle length. All other females showed a normal (regular) estrous cycle of 4 days. The incidence of irregular estrous cycle length
showed no relationship to the dose, and was therefore considered unrelated to treatment
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 adverse effects seen up to and including the highest dose level tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no

Dose Formulation Analyses

Small responses at the retention time of the test item were observed in the chromatograms of the control group formulations prepared for use in Week 1, Week 6 and Week 13. The maximum contribution to the formulation samples based on peak area was 0.011% taking the dilution factor into account. As the maximum contribution was only 0.011%, the control group formulations were still acceptable.

The concentrations analyzed in week 1 in the formulations of groups exposed to the test item were in agreement with target concentrations (i.e. mean accuracies 98% and 100%).

The concentrations analyzed in week 6 in the formulations of the low and the mid dose group were in agreement with target concentrations (i.e. mean accuracies 97%).

The mean accuracies of the low and the mid dose group formulations were above the target concentration (i.e. 191% and 175% of target). An ‘out of specification’ investigation did not reveal an error. Redilution of the formulation samples resulted in comparable mean accuracies (i.e. 199% and 179% of target). Based on the overall results in this study, these single out of spec results were considered not to have affected the overall conclusion of the study.

The formulations of the low, mid and the high dose groups were homogeneous (i.e. coefficient of variation ≤ 6.0%).

Conclusions:
An oral sub-chronic toxicity study was performed according to OECD/EC guidelines and GLP principles. No adverse effects were observed after 90-day repeated dosing to Dehyton DC, therefore the NOAEL was established to be 1000 mg/kg bw/day (based on solid content). This result is read across to the registered substance, the rationale is attached in Section 13.
Executive summary:

A sub-chronic toxicity study was performed according to OECD/EC guidelines and GLP principles. Wistar Han rats were orally exposed to Dehyton®DC by daily oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day for ninety days. Accuracy and homogeneity of formulations were confirmed by chemical analyses. No mortality occurred. A lower body weight compared to control was observed for males at 1000 mg/kg bw/day from the second week onwards with corresponding lower food consumption. In addition, slightly lower body weight compared to control with corresponding lower food consumption was also seen in males at 100 and 300 mg/kg bw/day starting in the second week. At the severity observed and in the absence of any histopathological effects, these effects were not considered to be adverse. At 100, 300 and 1000 mg/kg bw/day incidental effects related to breathing (abnormal breathing sounds, labored breathing, shallow breathing and increased and decreased respiratory rate) were noted in one or two animals per group on several days between Days 35 and 90. However, at the severity observed and in the absence of any histopathological effects, these effects were not considered to be adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, estrous cycle determination, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). No adverse effects were seen on reproduction parameters (estrous cycle length, spermatogenesis, weight, appearance and histopathology of reproduction organs).

Based on these results, the no-observed-adverse-effect level (NOAEL) for sub-chronic exposure was found to be 1000 mg/kg bw/day. This result is read across to the registered substance, the rationale is attached in Section 13.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was performed with a substance analogue, according to OECD/EC guidance and GLP principles (Klimisch 1 study). The rationale to read across the data is attached in Section 13.

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

The data for repeated dose toxicity are read across from a substance analogue. The rationale to read across the data is attached in Section 13.

Treatment with Amphoacetates C8-C18 (mono-acetate) 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 Amphoacetates C8 -C18 (mono-acetate) of 1000 mg/kg bw/day was established. For Amphoacetates C8-C18 (diacetate), there was a high mortality in the females (4/10) and one premature death in the males at 1000 mg/kg bw/day. 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. To follow-up the results seen with Amphoacetates C8-C18 (diacetate), a sub-chronic toxicity study was performed according to OECD guideline 408. In the sub-chronic study, the dosing volume was decreased compared to the 28 day repeated dose study in order to minimize risk of regurgitation. As in this study no mortality was seen, the loss of the high dose group in the 28 day repeated dose study was concluded to be indeed secondary to the test item. No toxicologically significant changes were noted in any of the parameters investigated in this study. No adverse effects were seen on reproduction parameters (estrous cycle length, spermatogenesis, weight, appearance and histopathology of reproduction organs). Based on these results, the no observed adverse effect level (NOAEL) for sub-chronic exposure was found to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data, the registered substance is not classified for repeated dose exposure according to CLP Regulation (EC) No. 1272/2008.