Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C17 odd-numbered, C17-unsatd. alkyl) derivs. and sodium hydroxide and chloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two screening studies for reproduction and developmental effects were performed according to OECD 422 with two representatives of the registered substance (a mono- and a diacetate form, respectively). 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Oct 2017 - 22 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: See below under Principles of method if other than guideline

Principles of method if other than guideline:

In addition, the procedures described in this study plan essentially conform to the following
guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.

GLP compliance:

yes

Limit test:

no

Justification for study design:

At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Species:

rat

Strain:

other: Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 275 - 307 g (males) and 198 - 241 g (females)
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were gr oup housed (up to 5 animals of the same sex and same dosing group together) in Macrolon cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon cages. During the post-mating phase, males were housed in Macrolon cages with a maximum of 5 males/cage. Females were individually housed in Macrolon cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 42-73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 04 Oct 2017 To 27 Nov 2017

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING FORMULATIONS
Test item dosing formulations (w/w) were homogenized by swirling to visually acceptable levels at appropriate concentrations to meet dose level requirements. Bulk formulations sufficient for one week of dosing were prepared once a week as clear colorless solutions. After homogenizing the bulk formulations to a visibly acceptable level, each (bulk) formulation was then divided into 7 aliquots for daily dosing and stored in the refrigerator protected from light (maximum storage in the refrigerator for 8 days after preparation). The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing for adjustment to room temperature.
On each day of use the test item dosing formulations were kept at room temperature until dosing. To avoid foam formation and optimal homogeneity, the dosing formulations were swirled shortly before use for dosing. An adjustment was made for specific gravity of the test item.

DOSE VOLUME
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. Detection of mating was not confirmed in first instance for one female. Apparently, mating was overlooked in the assessment of the vaginal lavage in first instance. The mating date of this animal was determined a few days later based on detection of sperm cells on the vaginal lavage. Consequently, this couple was separated 4 days after the actual mating date.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Concentration and homogeneity analyses were performed by using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) for each sampling time point were collected. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Samples were taken in week 1 for concentration determination (all groups) and homogeneity (low and high dose groups, the homogeneity results obtained from the top, middle and bottom for the low and
high dose group preparations were averaged and utilized as the concentration results.

Duration of treatment / exposure:

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 postcoitum, 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.

Frequency of treatment:

Once daily, 7 days per week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

A 10-days dose range finder was conducted to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. The test item and vehicle were administered to 3 females/group by single daily oral gavage for 10 days. Clinical Observations were done at least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body Weights: On Day 1 prior to dosing and on Days 5 and 10. Food Consumption: Over Days 1-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 10 after the last observation of clinical signs (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed. No signs of toxicity were noted at any dose level. Based on these results, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day.

Positive control:

No.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Time schedule: Twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Once daily
During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

BODY WEIGHT: Yes
Time schedule for examinations: Prior to first dosing, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION:
Yes. Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION:
Yes. Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOLOGIC OBSERVATIONS
No

HEMATOLOGY
Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.

CLINICAL CHEMISTRY
Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.
- Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).

URIANALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (LND 6-13).
- Tests were performed after completion of clinical observations (including arena observation).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: According to test guidelines. Hearing ability, pupillary reflex, static right ing reflex , fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

GENERAL REPRODUCTION DATA:
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of
copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

For the testes of all selected males (n=5) of the control and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation.

PARAMETERS EXAMINED
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities. Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the guidelines were prepared for microscopic examination and weighed, respectively.
For males that failed to sire and females that failed to deliver pups histopathological examination of cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina was performed.
All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
For the testes of all selected males of the control and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Postmortem examinations (offspring):

From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group; mid dose group vs. control group; high dose group vs. control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100
- Live birth index: (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100

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.

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2017 - 07 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY OF TEST MATERIAL
- Stability for at least 7 days in the refrigerator is confirmed over the concentration range 1.0 to 200
mg/mL
- Stability for at least 6 hours at room temperature under normal laboratory light conditions is conf
irmed over the concentration range 1.0 to 200 mg/mL
CORRECTION FACTOR
Purity/Composition correction factor: Yes, correction factor is 2.554 based on solid content.
No specific handling conditions required

Species:

rat

Strain:

other: Crl:WI(Han).

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.This animal model has been proven to be susceptible to the effects of reproductive toxicants. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks (males); 13 weeks (females).
- Weight at study initiation: 276-312 gr (males); 205-242 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
Lactation phase: females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity monitoring: animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 20 - 22.
- Humidity (%): 37 - 59.
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15-Aug-2017 to 07 Dec 2017

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 7 days as a solution, formulated in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item.

DOSE VOLUME
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After 14 days of unsuccessful pairing one female who at 100 mg/kg bw/day had not shown evidence of mating was separated from her male.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory:
- Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
- Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
- Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating and up to and including the day before 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). Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily, 7 days per week.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels were selected based on the results of a 11-day dose range finder with oral administration of Miranol Ultra C32 in rats. No guidelines were applicable as this study was intended for dose level selection purposes only.

Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination, organ weights and histopathology. Only females with live offspring were selected.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: Clinical observations were performed at least twice daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were performed immediately after dosing and 1 hour (± 15 minutes) after dosing.

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION:
Yes. Quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:No

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.
- Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (LND 6-13).
- Tests were performed after completion of clinical observations (including arena observation).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: According to test guidelines. Hearing ability, pupillary reflex, static righting reflex , fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group, the male that failed to sire and the male that died at 1000 mg/kg bw/day before termination of the experiment).

Litter observations:

STANDARDISATION OF LITTERS
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Blood samples were collected from two of the surplus pups for the thyroid hormone analysis.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, anogenital distance, areola/nipple retentionphysical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1, 4, 7 and 13 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 (externally) and at PND 14-16 (both externally and internally).

GROSS EXAMINATION OF DEAD PUPS
- Yes, if possible, defects or cause of death were evaluated.
- The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.
- If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS
- The organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of an organ of a pair was determined. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes
All tissues as defined in the guidance were examined histopathologically. For the testes of all selected males of the control and high dose groups, all males that failed to sire, and one male that died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Postmortem examinations (offspring):

SACRIFICE
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
- All remaining pups were euthanized on PND 14-16.

GROSS NECROPSY
- Descriptions of all external abnormalities were recorded.
- Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).Particular attention was paid to the external reproductive genitals to examine signs of altered development.


HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group; mid dose group vs. control group; high dose group vs. control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Post implantation survival index: (Total number of offspring born/Total number of uterine implantation sites) x 100

Offspring viability indices:

- Live birth index: (Number of live offspring on day 1 after littering/Total number of offspring born) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live offspring on day 4 before culling/Number live offspring on day 1 after littering) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/Number live offspring on day 4 (after culling)) x 100

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 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

Description (incidence and severity):

Relative Food Consumption calculated against the body weight for scheduled intervals

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 had slightly lower activated partial thromboplastin time (APTT) values than concurrent controls. As all values at 1000 mg/kg 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 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 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) 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 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 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 group which showed no evidence of mating. The irregular cycle noted in one female of the 1000 mg/kg 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%).

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).

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.

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 according to OECD/EC guidelines and GLP principles (Klimisch 1 studies).

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

Two screening studies for reproduction and developmental effects were performed according to OECD 422 with two representatives of 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.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2019 - 18 Jul 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Testing was initiated as requested in ECHA decision TPE-D-2114359620-51-01/F. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed in such a way that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity/Composition correction factor: 2.08 (based on solid content);
Stability in water for at least 6 hours at room temperature under normal laboratory light conditions was confirmed over the concentration range 1.0 to 200 mg/mL, and stability in water for at least 7 days in the refrigerator was confirmed over the concentration range 1.0 to 200 mg/mL, Charles River Project 518371.

Species:

rat

Strain:

other: Wistar Han

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 188 - 278 g
- The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating) at the test facility
- Fasting period before study: No
- Housing: Individually in Macrolon plastic cages (MIII type, height 18 cm)
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 36-53
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 January 2019 To: 07 February 2019

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. For the high dose group (1000 mL/kg bw/day) pure test item was used. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure (Charles River Study 518371). For verification of concentration and homogeneity duplicate sets of samples (approximately 500 mg) were analysed. Concentration results were regarded to be acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Homogeneity results were regarded to be acceptable if the coefficient of variation (CV) of concentrations was ± 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Charles River Study 518371) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on mating procedure:

Not relevant, females were mated at the breeder.

Duration of treatment / exposure:

15 days (from Day 6 to Day 20 post-coitum, inclusive)

Frequency of treatment:

Once daily

Duration of test:

Animals were sacrificed Day 21 post-coitum

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a Combined 28-day Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test (OECD 422) by oral gavage of Dehyton® DC in rats (Charles River Study 518366, summarizd in Section 7.5.1), the preliminary results of a 90-day study (Charles River Study 20164357, summarizd in Section 7.5.1). It was attempted to produce graded responses to the test item. To minimize the risk of regurgitation of the test item risk, it was decided to lower the dosing volume by administering undiluted test item to the high-dose group in the current study. The lower dose groups was treated with the same volume of formulations of the test item in water to exclude any dose volume related effects. Final dose volume was 1.796 mL/kg bw, the dose volume for each animal was based on the most recent body weight measurement. To minimize foam formation, the dosing formulations were not stirred continuously during dose administration, but were swirled shortly before dosing instead.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (for general health/mortality and moribundity)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, cage debris was examined to detect premature birth

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs
- The uterus and thyroid glands were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for the animal euthanized in extremis. Organ to body weight ratio (using the body weight on Day 21 post-coitum) was calculated.
- Histopathologic examination was performed on thyroid glands from all control and high dose animals.

OTHER:
- Blood was collected from all surviving females on day of sacrifice (rats not fasted o/n) to analyse Triiodothyronine (T3), Thyroid-Stimulating Hormone (TSH) and Thyroxine (T4) levels

Ovaries and uterine content:

Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
- The number of corpora lutea;
- The weight of the (gravid) uterus (not for animals found dead or sacrificed before planned necropsy);
- The number of implantation sites;
- The number and distribution of live and dead fetuses;
- The number and distribution of embryo-fetal deaths;
- The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.

Fetal examinations:

Gross external examination was performed of the dam that was sacrificed before planned necropsy to detect late resorptions, recognizable fetuses, or normal implantations in development..

Litters of females surviving to scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are regarded to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and its weight (not for fetuses of animals sacrificed before planned necropsy) was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.

The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (1972). The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique (1965). After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. All carcasses, including the carcasses without heads, were eviscerated, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (1926). Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing, this was concluded not to be of influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

The numbers of fetuses (litters) available for morphological examination were 229 (22), 229 (22), 214 (20) and 227 (22) in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. External examination was performed for all fetuses, visceral examination was performed for approximately half of the fetuses of all groups, and skeletal examination was performed for the other half of fetuses. Moreover, as during eviscerating of the fetuses prior to skeletal staining prominent visceral malformations were noticed in one fetus at 100 mg/kg bw/day and one fetus at 1000 mg/kg bw/day, that were both selected for skeletal examination, these fetuses were also subjected to a visceral examination. Besides that,
skeletal examination was added for one fetus at 100 mg/kg/day (A043-04) to further examine an externally noted malformation. One dam was sacrificed pre-term,the morphological examination of her fetuses was reported separately

Statistics:

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Body Weight Gains: Calculated against the body weight on Day 6 post-coitum;
Corrected Body Weight Gains: Body weight recorded on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus;
Relative Food Consumption: Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum;
Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites)/number of corpora lutea)*100;
Post-implantation loss (%): ((number of implantation sites - number of live fetuses)/number of implantation sites)*100.

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter)/(number of viable fetuses/litter))*100.

Historical control data:

Historical Control Data on Crl:WI(Han) (outbred, SPF-Quality), Gestation Day 21 from studies performed 2014 - 2018 are included in the report and attached in the summary.

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

The data per dose for all dose groups (and for individual rats) in tabular form are included as attached background material. Results of statistical analyses are indicated (where applicable). In addition, historical control data of fetal examinations at this test facility and with this rat strain are attached.

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.

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.

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).

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 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 Amphoacetates C8 -C18 (diacetate form) 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.

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.