Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The data for effects on reproduction are read across from a substance analogue. Two screening studies for reproduction and developmental effects were performed according to OECD 422 with two representatives of one analogues, 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. The rationale to read across the data is attached in Section 13.

Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
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):
Males: Salivation occurred in most males in the 1000 mg/kg bw/day dose group throughout the treatment period. One male showed flat posture, red discoloration on the nose and laboured respiration and rales shortly before early sacrifice on day 20. The clinical signs among the surviving males comprised occasionally laboured breathing, rales, piloerection and red discolouration or discharge on the nose, generally during the reproductive period only. Salivation occurred from time to time during the premating and reproductive periods in several males in the 300 mg/kg bw/day dose group. Single males had severe rales, piloerection and a red discoloration on the nose sporadically throughout the treatment periods (premating and reproductive). In the 100 mg/kg bw/day dose group, piloerection, a scab on the tail and salivation occurred in single males in this dose group only on the first day one of dosing (salivation) or during the reproductive period (piloerection, scab).
Females: Salivation occurred in most females in the 1000 mg/kg bw/day dose group throughout the treatment period. Four females died prematurely. The clinical signs among the surviving females comprised occasional rales and piloerection during the treatment period. Salivation occurred from time to time during the premating and reproductive periods (post coitum, lactation) in some females in the 300 mg/kg bw/day dose group. Single females had hunched posture and laboured respiration once during the study. One to two females had piloerection, alopecia, scabs (cervical region) and a wound (cervical region) during the reproductive period. In the 100 mg/kg bw/day dose group, salivation occurred sporadically in one or two females during the treatment period.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were five animals (one male and four females) treated at 1000 mg/kg bw/day that were found dead or were sacrificed in extremis. The male was sacrificed in extremis on study day 20. Prior to euthanasia, this male had a flat posture, red discoloration on the nose and laboured respiration, rales and salivation. It gained weight prior to death. There were no relevant macroscopic findings but microscopic findings included slight necrosis of the tracheal epithelial, which may be suggestive for a gavage-related procedural incident. However, it cannot be excluded that like in the females discussed below, the morbidity may have been caused by regurgitation secondary to the test item. All other males survived to scheduled euthanasia. Three females treated at 1000 mg/kg bw/day were prematurely euthanized, due to respiratory clinical signs and one was found dead (3 females before mating at day 12-16 and 1 female at day 26). One female was euthanized in extremis on study day 12. Prior to euthanasia this female had slight hunched posture, severe laboured respiration and rales, piloerection, moderate salivation and was severely pale. The female lost weight prior to death. One female was found dead on study day 15. This female showed rales on day 1 and salivation during treatment, but no other clinical signs prior to death. One female was euthanized in extremis on study day 26. Prior to euthanasia this female had piloerection and severe laboured respiration, rales, salivation and ptosis. One female was euthanized in extremis on study day 16. Prior to euthanasia this female had slight quick breathing, piloerection and severe rales, shallow respiration, salivation and ptosis. The female lost weight prior to death. Macroscopically the lungs were not collapsed which microscopically could be explained by trachea and lung lesions (up to marked degree) such as: acute inflammation (trachea and lung), ulceration/erosions of bronchial epithelium and trachea, and bronchial fibrosis and/or hyperplasia. In one female, foreign material was found within the tracheal lumen.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated males remained in the same range as controls over the treatment period in the 100 and 300 mg/kg bw/day dose groups. In the 1000 mg/kg bw/day dose group male body weights and body weight gain were reduced, achieving levels of statistical significance on Day 15 and 29 of treatment (Day 1 and 15 of mating, respectively), resulting in a 9% lower mean body weight at the end of treatment compared to concurrent controls. It was noted that one male at 1000 mg/kg bw/day lost weight over the first week of treatment, but recovered during the second week. In the absence of any clinical sign during the first week, no toxicological significance was attached to this finding.
Body weights and body weight gain of treated females remained in the same range as controls over the treatment period in the 100 and 300 mg/kg bw/day dose groups and in the 1000 mg/kg bw/day dose group until the early termination.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for the males and females. A low food consumption was recorded for one cage, containing five high dose males over the first week of treatment. One of the males in this cage had lost weight during this period and it was considered likely that this was accompanied by a lower food consumption which consequently affected the food consumption value for this cage.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg bw/day dose group males, a decreased number of reticulocytes was observed (28,2%), achieving a level of statistical significance when compared to controls. Furthermore, a slightly higher mean corpuscular haemoglobin concentration (MCHC) value (4.3%) was calculated from the red blood cell parameters, also achieving a level of statistical significance when compared to the MCHC value in controls. No toxicologically relevant changes were noted in the other haematological parameters in males at 1000 mg/kg bw/day and in all haematology parameters in males at 100 and 300 mg/kg bw/day. The haematology results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice) and those for the females of the other groups (including controls) for PND 14-16 (at lactation). The slightly increased values for red blood cell, reticulocyte and platelet counts, and corresponding changes in Red Blood Cell Distribution Width and red blood cell derived indices Mean corpuscular haemoglobin and Mean corpuscular volume, in 1000 mg/kg bw/day dose group females in comparison with controls, were likely due to the difference in their physiological status, rather than indicative of a treatment-related effect. Although historical control data representative for Day 14 post-coitum were not available, the results obtained in females at 1000 mg/kg bw/day in this study were all within the normal range and it was concluded that there were no (marked) changes in any of the haematology parameters that indicated a treatment-related effect at sacrifice on Day 14 postcoitum. No toxicologically relevant changes were noted in haematological parameters in females treated at 100 and 300 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Coagulation: High mean values for Prothrombin Time and Activated Partial Thromboplastin Time were observed in control males in comparison with the historical control data. Since the mean values in treated males were similar to the historical control data and they showed no dose-response relationship, no toxicological significance was attached to the statistical significances apparent for PT in the treated groups.
Clinical biochemistry parameters: Statistically significant reductions in Aspartate aminotransferase in the 100 and 300 mg/kg bw/day dose groups were considered to have occurred by chance. Since no dose-response was present and a decrease in this enzyme level in plasma is of no biological relevance, no toxicological significance was attached to this finding. The clinical biochemistry results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice after 4 weeks of treatment) and those for the females of the other groups (including controls) for PND 14-16 (at lactation, after 7-8 weeks of treatment in this type of studies). In the six early sacrificed females at 1000 mg/kg bw/day, the mean values for several parameters showed a statistically significant difference when compared to controls, comprising; Alanine aminotransferase, Alkaline Phosphatase, total protein, albumin, urea, cholesterol, potassium, calcium and inorganic phosphate. Although the available historical control ranges for the blood-value of these parameters were applicable for lactating females, the absolute blood-values in the early sacrificed, pregnant females at 1000 mg/kg bw/day were still within the historical control range of the laboratory. Therefore the changes were considered minimal and likely due to the difference in their physiological status, rather than indicative of a treatment-related effect. This assumption might also be supported by the fact that no treatment-related changes in the clinical biochemistry parameters were observed in males at 100 mg/kg bw/day after a similar treatment period of 4 weeks. In lactating females at 300 mg/kg bw/day statistically significantly lower levels for total protein and albumin were observed on PND 14-16. The mean values for these parameters were at the lower limit of the normal range of this laboratory. No treatment-related changes were observed in the other clinical biochemistry parameters in females at 300 mg/kg bw/day and in all clinical biochemistry parameters in females at 100 mg/kg bw/day. Serum levels of T4 in F0 males were considered not to be affected by treatment.Serum levels of T4 in F0 females was not measured.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment in the males up to 1000 mg/kg bw/day and in the females up to 300 mg/kg bw/day. No functional tests were performed in females at 1000 mg/kg bw/day, because of their early sacrifice. A large variation in motor activity, i.e. mean total movements and ambulations, was observed between the males the four dose groups. In the absence of a clear dose response relationship and since all activity was within the normal range (Historical control data period 2015-2017: Total movements males: mean: 3341, P5-P95:1734-5284, n=200. Ambulations: mean: 883, P5-P95: 293-1143, n=200) the differences between groups were considered not indicative of a relation to treatment. In tested females, the motor activity was similar between treated and control groups. Males and females of all tested groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations in females up to 1000 mg/kg bw/day. Test item-related microscopic findings after treatment with Dehyton® DC were limited to the thyroid gland of the 300 and 1000 mg/kg bw/day group males: An increased incidence and severity in follicular cell hypertrophy in the thyroid gland of males was recorded at 300 mg/kg bw/day group (3/5 at minimal degree and 1/5 at slight degree compared to 1/5 at minimal degree in the controls) and at 1000 mg/kg bw/day group (2/5 at minimal degree and 2/5 males at slight degree, compared to 1/5 at minimal degree.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females, except one 300 mg/kg bw/day dose group female, had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 300 mg/kg bw/day (with normal litter). Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was 1/10 couples of the controls, 2/10 couples treated at 100 mg/kg bw/day and 1/10 couples treated at 300 mg/kg bw/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this finding.
In all females treated at 1000 mg/kg bw/day that were euthanized at Day 27-28, early placental/fetal development sites (development around day 9-12) were observed.
Mating index was considered not to be affected by treatment. Precoital time was considered not to be affected by treatment. All females showed evidence of mating within 5 days. Number of implantation sites was considered not to be affected by treatment.
Fertility index was considered not to be affected by treatment. A fertility index of 90%, 80%, 100% and 100% was observed for the controls and 100, 300 and 1000 mg/kg bw/day treated females respectively. It should be noted that a fertility index could be calculated for only 7/10 females at 1000 mg/kg bw/day. Three females of this dose group were dead before mating could have occurred.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect seen up to 300 mg/kg bw/day
Remarks on result:
other: No high dose group (1000 mg/kg bw/day) could be assessed related to mortality seen at highest dose tested due to regurgitation of the formulations (secondary effect)
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Neuropathological findings:
not examined
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. Eight of 13 pups in one litter in the 300 mg/kg bw/day dose group had alopecia. As this finding was confined to a single litter it was considered not related to treatment. Alopecia is known to occur in pups and when it occurs within a litter is assumed to be genetic.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. The number of dead pups at first litter check was 0, 0, and 9 from three litters for the control, 100, and 300 mg/kg bw/day dose groups, respectively, resulting in a live birth index of 100, 100 and 92%. The low value in the 300 mg/kg dose group was considered to have occurred by chance, because 7 out of 9 dead pups belonged to one litter. One additional pup in this litter was missing on Day 2 postpartum. This pup had been noted as having less milk and a scab on its snout. Two additional pups in this litter that survived to planned necropsy also had a scab on the snout. The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. A viability index of 97%, 100% and 99% was observed for the controls and 100 and 300 mg/kg bw/day treated females, respectively. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. A lactation index of 100%, 100% and 99% was observed for the controls and 100 and 300 mg/kg treated females, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment. Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. Treatment up to 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen at 300 mg/kg bw/day
Remarks on result:
other: No developmental data available for the 1000 mg/kg bw/day group, because of sacrifice of females due to secondary effect of test item before possible delivery.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Analysis of dose preparations: The concentrations analysed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 97% and 103%). A small response at the retention time of the test item was observed in the chromatograms of the control group (vehicle) formulation was considered an analytical issue, rather than the presence of test item in the control formulation. In the worst case of a contribution of 4.8% to the low dose group formulation the accuracy of the formulations was still within acceptable limits. The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation 6.4%).

Summary results dose range finding study: No mortality was observed. At both dose levels, slight salivation was noted (occasional salivation immediately after dosing was seen in 1/3 females at 500 mg/kg bw/day, and in 3/3 at 1000 mg/kg bw/day). Piloerection was noted at several occasions after dosing in all three high dose females. Diarrhoea was observed in 1/3 female on Day 10 at 500 mg/kg bw/day, this was not observed in the higher dose group. Body weight development was normal in all dosed rats and no abnormalities were seen at necropsy. Liver and kidney weights were considered to be normal for all females.

Conclusions:
An oral Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening was performed according to OECD/EC guidelines and GLP principles. Based on the mortality observed at the high level dose due to regurgitation of the test item (secondary effect), the parental no-observed-adverse-effect level (NOAEL) of Dehyton® DC was established at 300 mg/kg bw/day. No reproduction toxicity was observed up to the highest dose level tested, therefore the reproduction NOAEL was found to be 1000 mg/kg bw/day. This value is read across to the registered substance. The rationale to read across the data is attached in Section 13.
Executive summary:

An oral Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening was performed according to OECD/EC guidelines and GLP principles. Wistar Han rats were treated with Dehyton®DC 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). The surviving females in this group were early terminated shortly after the fourth female died, i.e. on Day 14 post-coitum. These early sacrificed Group 4 females, were all pregnant of a normal number of foetuses, and did not show any direct test item-related morphological changes. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. No reproduction toxicity was observed up to the highest dose level tested, although only 6/10 females at 1000 mg/kg could be examined. Based on these results, the no-observed-adverse-effect level (NOAEL) was found to be 300 mg/kg bw/day and the reproduction NOAEL was found to be 1000 mg/kg bw/day. This value is read across to the registered substance. The rationale to read across the data is attached in Section 13.

Endpoint:
screening for reproductive / developmental toxicity
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, non-treatment-related
Description (incidence and severity):
Animals of the 1000 mg/kg group and, on only a few occasions, in animals of the 300 mg/kg 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 group (and in a single male and female of the 100 mg/kg 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.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male and two females died in the highest dose group. 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 low. 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. Mortality was considered to be associated to the dosing technique and not test-item related.
Body weight and weight changes:
effects observed, non-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 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, non-treatment-related
Description (incidence and severity):
Females in the dosing groups of 1000 mg/kg bw/day 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.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-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, non-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 examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
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
Other effects:
effects observed, non-treatment-related
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. During the treatment (premating) period, all females had regular cycles of four days. Extended di-estrus during pairing occurred in one female of the 100 mg/kg bw/day group which showed no evidence of mating. The irregular cycle noted in one female of the 1000 mg/kg bw/day group was not test item-related as it occurred prior to initiation of treatment.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
One male at 300 mg/kg bw/day showed tubular atrophy in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This male had no normal spermatogenic staging profile. For the other evaluated testes no indications for abnormal spermatogenesis were seen.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Seven couples had no offspring: 2/10 control couples (both females not pregnant); 2/10 couples at 100 mg/kg bw/day (one female without evidence of mating; one female not pregnant); 2/10 couples at 300 mg/kg bw/day (one female not pregnant; one female implantation sites only); 1/10 couples at 1000 mg/kg bw/day (one female not pregnant).
Key result
Dose descriptor:
NOAEL
Remarks:
Parental/ Reproduction
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:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs were seen only in pups of the 1000 mg/kg bw/day group that did not survive until scheduled sacrifice. The findings in the pups of one litter (cold, no milk in the stomach) were related to the moribundity of the dam, which was not test item-related.
The nature of the findings in a few other pups (pale appearance, cold, no milk in the stomach) remained within the range seen normally in pups that die within a few days after birth. These findings were therefore regarded as unrelated to treatment.
Treated pups that survived until scheduled sacrifice showed no clinical signs and the incidental findings in pups of the control group (alopecia, pale appearance) remained within the range considered normal for pups of this age.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring in day 1 after littering compared with the total number of offspring born was considered not to be affected by treatment. At 1000 mg/kg bw/day, seven pups (from 4 litters) were found dead at first litter check. The pup mortality in one of the litters was considered to be related to the moribundity of the dam (euthanized in extremis at PND 1; her moribundity was not test item-related. The number of dead pups in the other 1000 mg/kg bw/day litters was within normal limits and judged to be unrelated to treatment.
The same was true for the incidental mortality at 100 mg/kg bw/day (one pup from 1 litter).The number of live offspring on PND 4 compared with the number of live offspring on PND 1 was considered not to be affected by treatment. At 1000 mg/kg bw/day, a total of 15 pups (out of 4 litters) died at PND 1-2 (versus none in the control group; the difference was statistically significant). These pups went missing, presumably cannibalized, died spontaneously, or were euthanized in extremis (10 pups of one dam that were euthanized at PND 1). The moribundity of the dam, resulting in poor health of her pups, was not test item-related. Pup mortality in the other 1000 mg/kg bw/day litters remained within the range considered normal for pups of this age and was therefore considered to be unrelated to treatment. For the same reason the incidental pup mortality at the lower dose levels was regarded as unrelated to treatment (one pup out of one litter at dose 100 mg/kg bw/day, two pups out of two litters at dose 300 mg/kg bw/day).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were not affected by treatment. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 1000 mg/kg be/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be test item-related. Absence of milk in the stomach was noted in most of the pups found dead at first litter check. As absence of milk in the stomach is a normal finding in pups that die prematurely and the incidence among pups of surviving dams remained in the range considered normal for pups of this age, this macroscopic finding was regarded as unrelated to treatment. No other macroscopic findings were noted in pups that died prematurely. Pups that survived until scheduled sacrifice showed no abnormalities at macroscopic examination.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Repro/ development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to the highest dose level tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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 (1%) was noted. At 500 mg/kg bw/day, weight loss was found in one animal between Days 5-11 (3%), in the other two reduced weight gain (0 or 2%) was found. At 1000 mg/kg bw/day, weight loss was seen in one animal (1%), 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. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day. Clinical signs to be expected in the main study generally occurred 1 and 3 hours after dosing (piloerection) or immediately after dosing (salivation). Based on these findings, daily clinical observations in the main study were conducted at least immediately after dosing and 1 hour (±15 minutes) after dosing.

Conclusions:
In an oral OECD 422 screening study, the parental and reproductive NOAEL were derived to be 1000 mg/kg bw/day. No parental, reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). This value is read across to the registered substance. The rationale to read across the data is attached in Section 13.
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. 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 exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-56 days). 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. The NOAEL for reproduction was established to be 1000 mg/ kg bw/day. This value is read across to the registered substance. The rationale to read across the data is attached in Section 13.

Effect on fertility: via oral route
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 available studies are performed with a substance analogue, according to OECD/EC guidelines and GLP principles (Klimisch 1 studies). The rationale to read across the data is attached in Section 13.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The data for effects on reproduction are read across from a substance analogue. Two screening studies for reproduction and developmental effects were performed according to OECD 422 with two representatives of one analogues, 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. In addition, a prenatal developmental study was performed according to OECD guideline 414 and GLP principles. The rationale to read across the data is attached in Section 13.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose:
read-across source
Limit test:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted in animals treated up to 1000 mg/kg bw/day. Salivation was seen after dosing among animals treated at 300 and 1000 mg/kg bw/day with increasing incidence and severity (mild to moderate) with increasing dose levels. This finding was regarded as not toxicologically relevant, taking into account the nature of the effect and its time of occurrence (i.e. after dosing). It was regarded as a physiological response rather than a sign of systemic toxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was related to treatment with the test item. At 300 mg/kg bw/day, one female was euthanized in extremis on Day 16 post-coitum for animal welfare reasons as it presented with a severe protruding spine. During veterinary examination, the animal also showed signs of severe pain, including restlessness, fear, abnormal posture, hunched posture, abnormal gait, vocalization, thickened area of the back
and hypersensitivity to touch of the back area. At necropsy, the spine was noted to be misshapen in the abdominal region, corresponding to the findings at clinical observation and were therefore not related to treatment with the test item. No other abnormalities were noted. The animal was pregnant, and carried normal developing fetuses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level over the study period.
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly lower serum levels of thyroid stimulating hormone (TSH) were noted at 300 and 1000 mg/kg bw/day (0.76x and 0.74x of controls, respectively), however without reaching statistical significance and within the historical control range. This slight decrease was mainly caused by 4 animals in the control group, for which TSH serum levels were above the historical control range (Historical control data for pregnant Wistar Han rats TSH (μLU/mL); mean (P5-P95): 0.407 (0.1270-0.7600), n=52; Total T3 (ng/dL); mean (P5-P95): 64.7 (51.00-84.50) n=47; Total T4 (μg/dL); mean (P5-P95): 2.12 (1.430-3.090), n=50), data for this study are included as attachment. This slight difference in TSH levels was therefore regarded to be unrelated to the test-item. Serum levels of total T3 and T4 were regarded to be unaffected by treatment up to 1000 mg/kg/day.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weights and thyroid:body weight ratios of treated animals were similar to those of control animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were regarded to have arisen as a result of treatment.
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 the thyroid glands of 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.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The number of early resorptions slightly increased upon increasing dose levels. However, no statistical significance was reached and all values remained within historical control data. Therefore, this slight increase was regarded to be unrelated to the test item.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At 300 mg/kg bw/day, one female was not pregnant. The pregnancy rate was within the normal range that could be expected for rats of this age and strain, therefore, this single occurrence was regarded to be unrelated to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects observed up to and including the highest dose tested (1000 /kg bw/day)
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal body weight (both sexes) was unaffected by treatment up to 1000 mg/kg bw/day.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on external morphology following treatment up to 1000 mg/kg bw/day. At 100 mg/kg/day, two fetuses were externally malformed. One fetus had an omphalocele and another fetus had no lower jaw and had a cleft palate. Skeletal examination substantiated the findings of the latter fetus, whereby the lower jaw was anomalous and appeared to be only visible after staining. Due to the single occurrence and occurrence at the low dose level, these malformations were regarded to be of spontaneous origin. No external variations were observed in animals treated up to 1000 mg/kg bw/day.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related effects on skeletal morphology following treatment up to 300 mg/kg bw/day. Skeletal examination revealed four different malformations, in addition to the underlying malformations in the fetus discussed before at 100 mg/kg bw/day. Bent limb bones occurred in two control fetuses, and in two fetuses at 100 and 1000 mg/kg bw/day, respectively. In addition, one fetus had fused skull bones and a vertebral anomaly without associated rib anomaly. A vertebral anomaly occurred in a single fetus of the control and 100 mg/kg bw/day groups as well. At 100 mg/kg bw/day, one fetus was observed with sternoschisis. All malformations were also previously noted among historical control fetuses and due to the low incidences and group distribution, they were regarded not to be test item-related. Among skeletal variations, 7th cervical ossification site showed a remarkable increase at 1000 mg/kg bw/day. Mean litter incidences were 1.5%, 5.2%, 4.6% and 11.3% per litter in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at the high dose was not statistically significant, but was outside the historical control data range (Historical Control Data Prenatal Developmental Studies Rat GD21 includes 49 studies, performed between 2014 and 2018. In total 6219 (1070) fetuses (litters) were skeletally examined. 7th Cervical ossification site(s); mean (p5-p95): 3.8% (0.0-8.7%)). Therefore, a test item-related effect cannot be excluded. All other variations were either limited to the control group only or occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, these variations were regarded not to be test item-related.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Visceral malformations occurred in two fetuses at 100 mg/kg bw/day and one fetus each at 300 and 1000 mg/kg bw/day. At 1000 mg/kg bw/day, one fetus had a right-sided aortic arch, ventricular septum defect and no eyes. At 300 mg/kg bw/day, one fetus was also affected with a ventricular septum defect and was in addition observed with the absence of the ductus arteriosus, situs inversus and abnormal lung lobation. The latter two findings were also observed at 100 mg/kg bw/day in one fetus together with three cardiovascular malformations (ventricular septum defect, interrupted aortic arch and retro-esophageal ductus arteriosus). The other affected fetus at 100 mg/kg bw/day had abnormal lung lobation and transposition of the great vessels.
Although no dose-response was observed in any of the cardiovascular malformations, a test item-related effect could not be excluded, as in case of right-sided aortic arch the incidence was above historical control data range (Historical Control Data Prenatal Developmental Studies Rat GD21 includes 49 studies, performed between 2014 and 2018. In total 6234 (1071) fetuses (litters) were viscerally examined. Aortic arch right sided: mean (p5-p95): 0.0% (0.0-0.3%); summary incidence: 2 (2) fetuses (litters); Absence of the eye: mean (p5-p95): 0.2% (0.0-1.5%); summary incidence: 7 (7) fetuses (litters); Abnormal lobation of the lung: mean (p5-p95): 0.1% (0.0-0.8%); summary incidence: 4 (4) fetuses (litters); Situs inversus: mean (p5-p95): 0.2% (0.0-1.1%); summary incidence: 14 (14) fetuses (litters); Ventricular septum defect: mean (p5-p95): 0.0% (0.0-0.0); summary incidence: 1 (1) fetuses (litters)); (aortic arch right-sided: 0.9% at 1000 mg/kg/day; ventricular septum defect: 0.9% at 100, 300 and 1000 mg/kg/day) or were previously not observed in any of the control fetuses in studies performed at this Test Facility between 2014 and 2018 (interrupted aortic arch, retro-esophageal ductus arteriosus, absent ductus arteriosus and transposition of the great vessels).
The remaining malformations were regarded to be of spontaneous origin: the incidences of absence of the eyes (0.6% at 1000 mg/kg/day), situs inversus (0.6% and 1.3% at 100 and 300 mg/kg/day, respectively) and abnormal lobation of the lung (1.5% and 1.3% at 100 and 300 mg/kg/day, respectively) remained within historical control ranges or were only slightly above. In addition, no dose-related trend could be established for the latter two findings. Only one visceral variation (small supernumerary liver lobes) was observed in this study. At the low incidence it occurred, this was regarded not to be related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal ano-genital distance (both sexes) noted after treatment up to 1000 mg/kg/day.
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
visceral malformations
Remarks on result:
other: The incidence and severity of the effects do not increase dose-dependently, therefore it cannot be excluded that the effects are not related to test item exposure.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: cardiovascular
Description (incidence and severity):
Cardiovascular malformations (including ventricular septum defects) at 100, 300 and 1000 mg/kg bw/day, in 4 (4) fetuses (litters) in total. As the incidence and severity of the effects do not increase dose-dependently, it cannot be excluded that the effects are not related to test item exposure.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
yes
Conclusions:
A prenatal developmental toxicity study was performed in rats according to OECD/EC guideline 414 and GLP principles.The maternal No Observed Adverse Effect Level (NOAEL) for Dehyton® DC was determined to be at least 1000 mg/kg bw/day. No developmental NOAEL could be determined, as severe cardiovascular malformations were observed at all dose levels. As the incidence and severity of the effects do not increase dose-dependently, it cannot be excluded that the effects are not related to test item exposure. The data is read across to the registered substance.
Executive summary:

A prenatal developmental toxicity study was performed according to OECD/EC guideline 414 and GLP principles. Time-mated female Wistar Han rats were treated with Dehyton® DC from Day 6 to 20 postcoitum, inclusive, 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. Correct dosing was confirmed by chemical analyses showing that the formulations were homogeneous and that the concentrations were accurate. No treatment-related mortality occurred during the study period and no test item-related changes were noted in clinical appearance, body weight, food consumption, macroscopic examination, thyroid hormone levels, thyroid weights, microscopic appearance of the thyroids, and fertility parameters (pregnancy rate, numbers of corpora lutea and implantation sites, and pre-implantation loss). Visceral examination of fetuses revealed severe cardiovascular malformations at 100, 300 and 1000 mg/kg bw/day, in 4 (4) fetuses (litters) in total. At 1000 mg/kg bw/day, one fetus had a right-sided aortic arch, ventricular septum defect and no eyes. At 300 mg/kg bw/day, one fetus had a ventricular septum defect, absence of the ductus arteriosus, situs inversus and abnormal lung lobation. At 100 mg/kg bw/day, two fetuses were viscerally malformed: one fetus had abnormal lung lobation and transposition of the great vessels, and the other fetus presented itself with situs inversus, abnormal lung lobation, interrupted aortic arch (between right subclavian and right carotid), retro-esophageal ductus arteriosus and a ventricular septum defect. Although no dose-response was observed in any of the cardiovascular malformations, it cannot be excluded that there is a possible test item-related relationship taken the above in consideration. Mean litter incidences of a 7th cervical ossification site were 1.5%, 5.2%, 4.6% and 11.3% per litter in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at the high dose was not statistically significant, but was outside the historical control data range1. Therefore, a test item-related effect cannot be excluded for this type of skeletal variation. No test item-related changes were noted in litter size, post-implantation loss, sex ratio, fetal body weights, fetal ano-genital distance, external and skeletal malformations and variations. In conclusion, based on the results of this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for Dehyton® DC was determined to be at least 1000 mg/kg bw/day. No developmental NOAEL for Dehyton® DC could be determined, as severe cardiovascular malformations were observed at all dose levels. However, as the incidence and severity of the effects do not increase dose-dependently, at this point it cannot be excluded that the effects are not related to test item exposure. The data is read across to the registered substance.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A prenatal developmental study was performed with a substance analogue, according to OECD/EC guidance and GLP principles (Klimisch 1 study). This data is read across to the registered substance.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproduction toxicity

Two screening studies for reproduction and developmental effects were performed according to OECD 422 with two representatives of one analogue, Amphoacetates C8-C18. In both studies, no reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in the reproductive parameters examined in both studies (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). For the study with the diacetate Amphoacetate C8-C18, the reproduction parameters were assessed for all groups, but the developmental effects could not be determined in the high dose group. In neither study developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day or 300 mg/kg bw/day). No treatment-related changes were noted in the developmental parameters investigated in the studies (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 14-16) and macroscopy). Furthermore, no effects on weight, macroscopy or histopathology were seen in the sub-chronic study on male and female reproductive organs performed with substance analogue diacetate Amphoacetate C8-C18. 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. Taken together, there are no indications that the registered substance has an adverse effect on reproduction.

Developmental toxicity

A prenatal developmental toxicity study was performed according to OECD/EC guideline 414 and GLP principles. Time-mated female Wistar Han rats were treated with Dehyton® DC from Day 6 to 20 postcoitum, inclusive, 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. Correct dosing was confirmed by chemical analyses showing that the formulations were homogeneous and that the concentrations were accurate. No treatment-related mortality occurred during the study period and no test item-related changes were noted in clinical appearance, body weight, food consumption, macroscopic examination, thyroid hormone levels, thyroid weights, microscopic appearance of the thyroids, and fertility parameters (pregnancy rate, numbers of corpora lutea and implantation sites, and pre-implantation loss). Visceral examination of fetuses revealed severe cardiovascular malformations at 100, 300 and 1000 mg/kg bw/day, in 4 (4) fetuses (litters) in total. At 1000 mg/kg bw/day, one fetus had a right-sided aortic arch, ventricular septum defect and no eyes. At 300 mg/kg bw/day, one fetus had a ventricular septum defect, absence of the ductus arteriosus, situs inversus and abnormal lung lobation. At 100 mg/kg bw/day, two fetuses were viscerally malformed: one fetus had abnormal lung lobation and transposition of the great vessels, and the other fetus presented itself with situs inversus, abnormal lung lobation, interrupted aortic arch (between right subclavian and right carotid), retro-esophageal ductus arteriosus and a ventricular septum defect. Although no dose-response was observed in any of the cardiovascular malformations, it cannot be excluded that there is a possible test item-related relationship taken the above in consideration. Mean litter incidences of a 7th cervical ossification site were 1.5%, 5.2%, 4.6% and 11.3% per litter in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The increase at the high dose was not statistically significant, but was outside the historical control data range1. Therefore, a test item-related effect cannot be excluded for this type of skeletal variation. No test item-related changes were noted in litter size, post-implantation loss, sex ratio, fetal body weights, fetal ano-genital distance, external and skeletal malformations and variations. In conclusion, based on the results of this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for the test item was determined to be at least 1000 mg/kg bw/day. No developmental NOAEL for Dehyton® DC could be determined, as severe cardiovascular malformations were observed at all dose levels. However, as the incidence and severity of the effects do not increase dose-dependently, at this point it cannot be excluded that the effects are not related to test item exposure. The data is read across to the registered substance.

Justification for classification or non-classification

Based on the available data, the registered substance is not classified for reproduction toxicity and effects on development according to CLP Regulation (EC) No. 1272/2008.