Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with Alkaterge E according to OECD Guideline No. 422, no reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, and number of implantations, estrous cycle, spermatogenic profiling (i.e. stage aware evaluation), and histopathological examination of reproductive organs). The parental and the reprodutction NOAEL in this study was 1000 mg/kg.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017 - April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification: Alkaterge E
Appearance: Brown viscous liquid
Batch: D598F47BC1
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 05 April 2020 (retest date)

Additional information
Test Facility Test Item Number: 208794/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name: 4-Ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol
CAS number: 68140-98-7
Molecular formula: C23H43NO2
Molecular weight: 365.60
Specific gravity / density 0.925 (25°C)
Stability in corn oil (vehicle): Stability for at least 5 hours at room temperature under normal laboratory light conditions and for at least 12 days in the refrigerator.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Female Crl: WI(Han) and male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 10 weeks old and weighed between 277 and 309 g, and females were 13 weeks old and weighed between 204 and 233 g.
Sex:
male/female
Details on test animals and environmental conditions:
1. Animal Identification
Prior to start of the pre-test period (females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pre-test period, reserve females were numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment received identification by earmark and tattoo. Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

2. Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pre-test period (females) or 8 days before the commencement of dosing (males).

3. Selection, Assignment, Replacement, and Disposition of Animals
A total of 40 females was selected at randomization before initiation of the pre-test phase. Any selected female without a regular oestrous cycle at the end of the pre-test phase was replaced by one of the 8 additional females having regular oestrous cycles. A total of 40 females with regular oestrous cycles continued in the study. The supernumerary females were removed from the study and their oestrous cycle results were kept in the raw data but were not reported. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately. Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

4. Housing
On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the 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. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities. Each cage was clearly labelled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

5. Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 20°C with an actual daily mean relative humidity of 47 to 71%. A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

6. Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

7. Water
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.
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. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. All samples were stored on dry ice immediately after sampling. All samples to be analyzed were shipped on dry ice to ABL BV (the next day). The analytical laboratory was notified before shipment of the samples. Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ -70 °C until analysis. Analyses were performed by using a validated analytical. Duplicate sets of samples (approximately 500 mg) for each sampling time point 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. Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. 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 (Test Facility Study No. 519525; Test Site Study No. ABL17248). Stability for at least 5 hours at room temperature under normal laboratory light conditions and for at least 12 days in the refrigerator is confirmed over the concentration range 1 mg/mL (1.09 mg/g) to 200 mg/mL (217 mg/g) (solution), Test Facility Study No. 519525; Test Site Study No. ABL17248.
Duration of treatment / exposure:
Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-55 days, i.e. 14 days prior to mating (with the objective to cover at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41, 51 or 54 days.
Details on study schedule:
The study was conducted with 10 animales per sex per dose. Five animals/sex/group were selected for functional tests, clinical pathology, macroscopic examination, organ weights and histopathology. The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-55 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrousoestrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41, 51 or 54 days. The first day of dosing was designated as Day 1.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
The study was conducted with 10 animales per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
A total of 40 females was selected at randomization before initiation of the pre-test phase. The study was conducted with 10 animales per sex per dose. Five animals/sex/group were selected for functional tests, clinical pathology, macroscopic examination, organ weights and histopathology. Any selected female without a regular oestrous cycle at the end of the pre-test phase was replaced by one of the 8 additional females having regular oestrous cycles. A total of 40 females with regular oestrous cycles continued in the study. The supernumerary females were removed from the study and their estrousoestrous cycle results were kept in the raw data but were not reported. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately. Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
Positive control:
Not applicable
Parental animals: Observations and examinations:
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. Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.

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.

During the first week of dosing, these observations were performed 1 hour (±15 minutes) after dosing. This was based on the peak effect of occurrence of clinical signs observed in the dose range finder (Test Facility Study No. 519524, see Appendix 6). However, since salivation was noted for a few animals in Groups 2, 3 and 4 after dosing on 17 October 2017 (treatment Day 7) (taken from study daybook), an extra observation 0-15 minutes after dosing was introduced starting on treatment Day 8. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

F0-Generation: clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted 1 hour (± 15 min) post-dose. Animals were weighed individually on the first day of treatment (prior to 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 terminal weight was recorded on the day of scheduled necropsy. 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.

Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed at 1 hour (± 15 min) after dosing, after completion of clinical observations. The following tests were performed (abbreviations mentioned in the respective tables are indicated between brackets):
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).
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 finefiner movements like grooming, weaving or movements of the head.


Oestrous cyclicity (parental animals):
Oestrous 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 (pretestpre-test period) to ensure that all females had regular oestrous cycles at start of treatment. For one female (group 2), no vaginal lavage was performed on pretestpre-test Day 2 as she was noted with red staining in the vaginal region. At the end of the pre-test phase she was classified as not having regular oestrous cycles. Therefore, she was replaced before initiation of dosing by one of the 8 additional females having regular oestrous cycles. For all females, daily vaginal lavage continued during 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 oestrus.
Sperm parameters (parental animals):
For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative histopathological examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
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.
Postmortem examinations (parental animals):
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 surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:

Males (which sired and failed to sire): following completion of the mating period (a minimum of 28 days of administration). All males surviving to scheduled necropsy were fasted before their scheduled necropsy. Water was available.

Females which delivered: PND 14-16.
Females which failed to deliver: with evidence of mating, post-coitum Day 25 (nos. 45 and 68).
without evidence of mating: 27 days after the last day of the mating period (no. 52).

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. Selected organs (see report) weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for one female that died after blood sampling on the day of necropsy. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Representative samples of seletected tissues were collected from all animals and preserved in 10% neutral buffered formalin. The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- selected animals and unscheduled deaths: tissues identified in table 10 in the report (except animal identification), aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- males that failed to sire (except for males which were selected) and females that failed to delivery pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- remaining animals: gross lesions/masses.

All tissues as defined under histology – F0-Generation (section 4.12.6) in report were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. For the testes of all selected males of Groups 1 and 4, 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):
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination. 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. 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. 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 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 matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
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.

Incidence
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:
Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females.

- Mating (%): 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 mated (x 100)
- Gestation index (%): Number of females with living pups on Day 1 / 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:
Group mean values were calculated from individual litter values.

- 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, treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality related to Alkaterge E occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain 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):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. When compared to the concurrent control group, a slightly, but statistically significantly higher mean value for absolute food intake was recorded for females in Group 3 from Days 14-17 post-coitum. This observation was considered to be unrelated to treatment since no trend was apparent regarding dose, and in case of toxicity a decrease rather than increase would have been expected.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters. The statistically significant lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) that occurred in the 300 mg/kg males were considered unrelated to treatment because neither reduction was dose-dependent.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical biochemistry parameters distinguished animals in the high dose group (1000 mg/kg) from concurrent control animals:
• Higher alanine-aminotransferase (ALAT) in males and females (relative difference from control: 3.88% fold and 1.64 fold%, respectively).
• Higher aspartate-aminotransferase (ASAT) only in males (relative difference from control: 1.52 fold%).
• Higher urea only in males at 1000 mg/kg (relative difference from control: 1.34 fold%).

The statistically significantly higher potassium for males at 1000 mg/kg (relative difference from control: 115%) was not considered to be related to treatment. Changes compared to the concurrent control group were only slight and there were no supportive haematological or histopathological changes (such as renal findings or evidence for haemolysis). All remaining clinical biochemistry parameters in treated animals remained in the same range as for the concurrent controls.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In males of the 1000 mg/kg group, mean grip strength of the hind legs was approximately 12% lower compared to the mean value of the concurrent control group (not statistically significant). The average value (653 gram) was within the range of the available historical control data and was therefore considered not to be toxicologically relevant. Moreover, there were no corroborating changes in foreleg grip strength or other end points in the neuromuscular domain. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Some females at 1000 mg/kg showed a higher activity, both in terms of total movements and ambulations throughout the entire motor activity testing session of 1 hour (relative difference from control: 140% and 161%, respectively). No toxicological significance was attached to this finding as average values for total movements (4521) and ambulations (1226) did not reach statistical significance when compared to the concurrent controls and remained within the normal range of biological variation. Moreover, motor activity habituation profile of these high dose females appeared similar to those of other groups. In addition, there were no other changes in functional observation parameters or clinical appearance that would support this observation. As such, this variation in motor activity of high dose females was not considered to represent a toxicologically relevant effect. Some females at 1000 mg/kg showed a higher activity, both in terms of total movements and ambulations throughout the entire motor activity testing session of 1 hour (relative difference from control: 140% and 161%, respectively). No toxicological significance was attached to this finding as average values for total movements (4521) and ambulations (1226) did not reach statistical significance when compared to the concurrent controls and remained within the normal range of biological variation2. Moreover, motor activity habituation profile of these high dose females appeared similar to those of other groups. In addition, there were no other changes in functional observation parameters or clinical appearance that would support this observation. As such, this variation in motor activity of high dose females was not considered to represent a toxicologically relevant effect.
Immunological findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Alkaterge E were noted in the thyroid gland, duodenum/jejunum of males and the thymus of females at 1000 mg/kg. In the thyroid gland of high dose males (1000 mg/kg), an increased incidence and severity (up to a slight degree) of follicular cell hypertrophy and colloid alteration was present. In the duodenum/jejunum of high dose males (1000 mg/kg), mucosal vacuolation (up to a slight degree) was present. In the thymus of high dose females (1000 mg/kg), lymphoid atrophy (up to a slight degree) was present. The remainder 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.

There were 1/10 couples of the control group, 1/10 couples at 100 mg/kg and 1/10 coupes at 300 mg/kg which failed to deliver pups. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. 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.

Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrousoestrous cycle were not considered to have been affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in female nos. 52, 68 and 77 in the 100, 300 and 1000 mg/kg dose groups, respectively, and female no. 55 in the 100 mg/kg dose group had an irregular cycle (i.e. one cycle of 6 days). Female no. 52 was not mated and female no. 68 was not pregnant, but female nos. 55 and 77 had normal litters. Given their incidental nature, absence of a dose-related incidence and/or absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was not considered to be affected by treatment. All females except for one female in the 100 mg/kg dose group showed evidence of mating. Precoital time was not considered to be affected by treatment. Most females mated within 4 days. One female in each of the 300 and 1000 mg/kg dose groups mated during the second week of the cohabitation period (Days 12 and 13, respectively). This slightly delayed mating is still within the normal range of biological variation. In the absence of a dose-related incidence, it was considered to be unrelated to treatment. Number of implantation sites were comparable in all groups and within the expected range. Fertility index was not considered to be affected by treatment.

The fertility index was 90% in the control group, and 100%, 90% and 100% for females in the 100, 300, and 1000 mg/kg dose groups, respectively. One female each in the control group (no. 45) and 300 mg/kg group (no. 68) were not pregnant. Since these single cases of non-pregnancy showed no dose-related incidence across the dose groups, occurred in absence of any reproductive toxicity and remained within the range considered normal for rats of this age and strain, this was not considered to be related to treatment.

Gestation index and duration of gestation were not affected by treatment. All pregnant females delivered living pups.

Key result
Dose descriptor:
NOAEL
Remarks:
parental effects
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse findings up to the highest dose level tested (1000 mg/kg).
Key result
Dose descriptor:
NOEL
Remarks:
parental effects
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No paretal toxicity was observed
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).
Ophthalmological findings:
not examined
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were related to treatment. The nature and incidence of observed clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. The post-implantation survival index was 86% for the control group and 91%, 96% and 93% for females in the 100, 300, and 1000 mg/kg dose groups, respectively. For females 46 (Group 1) and 67 (Group 3), the numbers of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the (14-15) days of) lactation. No toxicological relevance was attached to this finding in the current study. The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. One pup each in the control and 300 mg/kg groups were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. Five pups of the control group and two pups at 100 mg/kg were missing or found dead on PND 2 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to the dead/missing pups in the 100 mg/kg group since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups at 1000 mg/kg (both sexes) were lower on PND 7 and PND 13 as compared to the concurrent control group (relative difference decrease from control: 8911% and 8416% for combined pup weight, respectively). At the end of the observation period (PND 13), mean body weights of male and female pups (26.1 and 25.4 gram, respectively) were at the low end of the available historical control range5. Based on this and the magnitude of the change (16%), it was considered to be an adverse effect. Body weights of pups in the lower dose groups of 100 and 300 mg/kg were not considered to be affected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Description (incidence and severity):
Serum levels of total T4 in male and female PND 13-15 pups were not considered to be affected by treatment.
Urinalysis findings:
not examined
Description (incidence and severity):
Sex ratio was not considered to be affected by treatment. Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment. Treatment up to 1000 mg/kg 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. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Serum levels of total T4 in male and female PND 13-15 pups were not considered to be affected by treatment.
Body weights of pups at 1000 mg/kg (both sexes) were statistically significantly lower on PND 7 and PND 13 as compared to the concurrent control group (relative decrease from control: 11% and 16% for combined pup weight, respectively). Pup body weights at PND 1 were similar across the groups, indicating that the observed lower weights from PND 7 onwards resulted from reduced post-natal growth. No treatment-related changes were noted in any of the other developmental parameters investigated in this study including: gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels (PND 14-16 pups) and macroscopic examination.
Key result
Dose descriptor:
LOAEL
Remarks:
developmental effects
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
developmental effects
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Conclusions:
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). Pup body weights at PND 1 were similar across the groups, indicating that the observed lower weights from PND 7 onwards resulted from reduced post-natal growth. No treatment-related changes were noted in any of the other developmental parameters investigated in this study including: gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels (PND 14-16 pups) and macroscopic examination. Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the no-observed-adverse-effect level (NOAEL) of Alkaterge E were 1000 mg/kg bw fpr parental effects and reproductive effects. The developmental NOAEL was 300 mg/kg , based on reduced growth of pups in the 1000 mg/kg group.
Executive summary:

The objectives of this study were to determine the potential toxic effects of Alkaterge E when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg bw, based on the results of the dose range fnder.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs,functional observations (for 5 selected animals/sex/group),body weight and food consumption, estrous cycle determination,clinical pathology (for 5 selected animals/sex/group),measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

No adverse parental effects were observed up to the highest dose level tested (1000 mg/kg).Non-adverse, treatment related effects included, a dose-related increase of slight to moderate salivation in all treated groups approximately 1 hour after dosing, changes in clinical biochemistry parameters related to treatment at 1000 mg/kg that included higher values for ALAT (both sexes), ASAT and ALP (males only). In addition, a statistically significantly higher liver weight (relative to body weight) in males treated at 1000 mg/kg (relative difference from control: 119%). In the absence of any histopathological correlate, these findings were not considered to be adverse. No treatment-related or toxicologically relevant changes were noted in any of the other parental parameters investigated in this study including: mortality, clinical appearance, body weight, food consumption, functional tests, haematological investigations, thyroid hormone (T4) analyses, macroscopic examination and organ weights

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, and number of implantations, estrous cycle, spermatogenic profiling (i.e. stage aware evaluation), and histopathological examination of reproductive organs). Body weights of pups at 1000 mg/kg (both sexes) were statistically significantly lower on PND 7 and PND 13 as compared to the concurrent control group (relative difference from control: 89% and 84% for combined pup weight, respectively). Pup body weights at PND 1 were similar across the groups, indicating that the observed lower weights from PND 7 onwards resulted from reduced post-natal growth. No treatment-related changes were noted in any of the other developmental parameters investigated in this study including: gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels (PND 14-16 pups) and macroscopic examination.


Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Alkaterge E were established:

Parental NOAEL:            1000 mg/kg

Reproduction NOAEL:    1000 mg/kg

Developmental NOAEL:    300 mg/kg (based on reduced growth of pups in the 1000 mg/kg group).

 

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:
A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with Alkaterge E according to OECD Guideline No. 422 and under GLP.
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

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with Alkaterge E according to OECD Guideline No. 422, body weights of pups at 1000 mg/kg (both sexes) were statistically significantly lower on PND 7 and PND 13 as compared to the concurrent control group (relative difference from control: 89% and 84% for combined pup weight, respectively). Pup body weights at PND 1 were similar across the groups, indicating that the observed lower weights from PND 7 onwards resulted from reduced post-natal growth. No treatment-related changes were noted in any of the other developmental parameters investigated in this study including: gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels (PND 14-16 pups) and macroscopic examination.

The parental NOAEL in this study was 1000 mg/kg. The developmental NOAEL was 300 mg/kg (based on reduced growth of pups in the 1000 mg/kg group).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with Alkaterge E according to OECD Guideline No. 422 and under GLP.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the OECD 422 study, Alkaterge E should not be classified for reproduction toxicity. No adverse effects were observed up to the highest dose (1000 mg/kg bw) tested.