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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422 study
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
Details on route of administration:
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.
Vehicle:
corn oil
Details on oral 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.
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.

Accuracy no test item was detected in the Group 1 formulation. The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
Homogeneity: the formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
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.
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:
10 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
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.
Positive control:
not applicable
Observations and examinations performed and frequency:
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.

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.
Sacrifice and pathology:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. 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.
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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. A dose-related increase of slight to moderate salivation was seen in all treated groups. This was first observed on Day 7 of treatment approximately 1 hour post-dose (taken from study daybook). With the introduction of an extra observation immediately after dosing, it could be demonstrated that salivation was an immediate response to dosing. Therefore, it was considered to be a transient, physiological response rather than a sign of systemic toxicity.
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. 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
Description (incidence and severity):
Serum level of total T4 in F0-males was considered to be unaffected by treatment. The slightly, but statistically significantly lower serum level of total T4 in F0-males of the 300 mg/kg group was considered unrelated to administration of the test item as mean value was within the historical control data recorded for males of this age and strain and observed change occurred in absence of a dose-related trend.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in heart weight (absolute and relative to body weights) was noted in the 300 and 1000 mg/kg/day group males and a statistically significant increase in liver weights (relative to body weights) was noted in males at 1000 mg/kg/day. The lower heart weight in males treated at 300 and 1000 mg/kg was most likely the result of a relatively high heart weight (absolute and relative to body weight) recorded in the control males. As heart weights in all groups were within the historical control data5 recorded for males of this age and strain and observed weight changes occurred in absence of a dose-related trend and a histopathological correlate, this finding was regarded unrelated to treatment with the test item. The statistically significantly higher liver weight (relative to body weight) in males treated at 1000 mg/kg (119% increase compared to the controls) was considered to be treatment-related as individual values were outside the available historical range, but not adverse due the absence of a histopathological correlate. There were no other test item-related organ weight changes. The lower uterus weight (absolute) in females at 1000 mg/kg (relative difference from control: 78%) was considered not to be test item-related as individual values were within the historical control data7 recorded for females of this age and strain and the observed weight change occurred in absence of a histopathological correlate. In addition, all females in the high dose group had normal litters.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A macroscopic finding considered to be related to the treatment with the test item was present in the thymus: a reduced size was recorded in a single female at 1000 mg/kg (microscopic correlate: lymphoid atrophy). Other findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.
Neuropathological findings:
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.

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
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Organ:
salivary glands
Conclusions:
In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) with Alkaterge E, no adverse parental effects were observed up to the highest dose level tested (1000 mg/kg).
Executive summary:

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. 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 finder.

 

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, oestrous 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, pre-coital 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, pre-coital time, and number of implantations, oestrous 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 NOEL:                  300 mg/kg

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).

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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