Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-02 to 2018-07-13
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
see Overall Remarks / Attachments
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: Sodium oleylamphopolycarboxyglycinate
Chemical Name: active ingredient: Sodium oleylamphopolycarboxyglycinate
Old CAS No.: active ingredient: 97659-53-5
New CAS no: 2060541-49-1
Batch No.: 1604972
Physical State: liquid
Molecular Weight: 838.87 g/moL
Active Ingredient: 30.1 % (active is corrected for sodium chloride solids)
Date of Production: 23 August 2017
Storage Conditions: room temperature
Expiry Date: 23 August 2019


Specific details on test material used for the study:
Name: Sodium oleylamphopolycarboxyglycinate
Product: AMPHOLAK XO7/C
Chemical Name: active ingredient: Sodium oleylamphopolycarboxyglycinate
CAS No.: active ingredient: 97659-53-5
Batch No.: 1604972
Physical State: liquid
Molecular Weight: 838.87 g/moL
Active Ingredient: 40.5 % (was taken into account for final formulation)
Date of Production: 23 August 2017
Storage Conditions: room temperature
Expiry Date: 23 August 2019
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 342 - 402 g (mean: 373.50 g, ± 20 % = 298.80 – 448.20 g)
females: 212 - 257 g (mean: 233.28 g, ± 20 % = 186.62 – 279.93 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.


Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Nesting material were provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions


Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
Each animal was marked with its identification number by individual ear tattoo or tail marking.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua ad iniectabilia (sterile water)
Remarks:
Manufacturer: Deltamedica, Batch No.: 612118 and 702240, Expiry Date: 11/2019 and 01/2020
Details on oral exposure:
According to the results of a previous dose range finding study (BSL Munich Study No. 163485, non GLP) and in consultation with the sponsor the following doses were selected for the three dose groups (LD = low dose, MD = medium dose, HD = high dose) and one control group (C).
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 69 days in females, i.e. during 20 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 37-38 days was completed.
Inadvertently for first 5 days (6 to 10 November 2017), old batch of test substance was used and therefore after discussion with sponsor, it was decided to increase premating period from originally planned 14 days to additional 5-7 days so that there was 14 days premating period with new batch test item administration.
Inadvertantly, the last remaining animal in the study (female LD No. 54) was not dosed the day before necropsy (PND 11).


Control: 0 mg/kg/d
Low Dose: 100 mg/kg/d
Medium Dose: 300 mg/kg/d
High Dose: 1000 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.


The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 162887). Based on this study the formulation was proved to be a true solution and the repetition of homogeneity measurement in the main study was not necessary.
Samples were taken for substance concentration from the middle of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 162888) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.
The phase plan was amended to the study plan. The results are reported in the appendix of the final report.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 69 days in females, i.e. during 20 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 37-38 days was completed.
Inadvertently for first 5 days (6 to 10 November 2017), old batch of test substance was used and therefore after discussion with sponsor, it was decided to increase premating period from originally planned 14 days to additional 5-7 days so that there was 14 days premating period with new batch test item administration.
Inadvertantly, the last remaining animal in the study (female LD No. 54) was not dosed the day before necropsy (PND 11).
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
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:
100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
After the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.
During the study all animals were visually monitored to see if there is condition of polydipsia.


Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and only during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).


Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.


Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals (10.20).
Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined:
haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso) and large unstained cells (Luc).

Blood Coagulation
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT) and activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined:
alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP),
albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K).
From 2 female pups/litter on day 4 after birth from all dams and 2 pups/litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).
Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary based on the fact that no histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups. Other hormones were not measured. Pup blood was pooled by litter for thyroid hormone analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated when litter size dropped below 8 pups. Whenever there was only one pup available above a litter size of 8, only one pup was sacrificed on PND 4.


Urinalysis
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded.
The following parameters were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL):
specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood and leukocytes.


Sacrifice and pathology:
Pathology
All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 37-38 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy (except for animal no. 59, 70, 73, 75) to determine the stage of estrous cycle.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The wet weight of the organs [testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), uterus with cervix, ovaries (paired weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) – weighed after fixation (complete weight), thymus, liver, spleen, kidneys (paired weight), brain, adrenal (paired weight), heart and pituitary gland] of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead were not recorded. Reproductive organs were weighed in all animals.
Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

Adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes, Harderian glands, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric), lymph nodes (axillary), mammary gland area (male and female), oesophagus,optic nerves, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina of the same selected animals from each group were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70% ethanol.
All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.
Thyroid/parathyroid glands from non-selected adult animals were preserved for potential histopathological examination. The thyroid gland weight was measured after fixation.


Histopathology
A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. . Histopathological examination of thyroid gland from pups and from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males and pups sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in any organ/tissue of the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

Statistics:
A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and litter data were performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results
Mortality:
mortality observed, non-treatment-related
Description (incidence):
see Details on results
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see Details on results
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):
see Details on results
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see Details on results
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see Details on results
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
see Details on results: Thyroid Hormone (T4) Analysis
Details on results:
Clinical Observations
In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were moderate salivation and moving the bedding on one day in 1 LD male and on few days in one MD male. Moving the bedding in all HD males and slight to moderately increased salivation in 2 HD males was also observed on few days during premating day 1 to mating and post mating day 14. Abnormal breathing in 2 HD males was observed between premating day 2-10.
In terminally sacrificed females, major clinical signs observed during the treatment period (premating day 1 to PND 12) were moving the bedding in 3 MD (on few occasions between PMD 17 - PND 12) and in all HD animals (between PMD 1 - PND 12 on majority days). A slight to moderately increased salivation was observed in 2 HD group females (few days between PMD 19 and PND 12). There was also moderate piloerection in one MD female on PND 0, slightly reduced spontaneous activity in one LD female on PMD 6 and in 2 HD females (No. 76 on PMD 1 and No. 71 on PMD 1-5 and 9-10), ataxia in one HD female No. 71 on PMD 1-5 and 9-10, abnormal breathing in one HD female on PND 4 and regurgitation of a part of the test item in one MD and HD female (No. 74 on GD 16 of the HD group, No. 62 on PND 11 of the MD group) observed.
On PMD 8, all HD all males and 9 HD female animals were observed with diarrhea. There were also low incidences of the clinical signs like alopecia on various body parts of the one HD male, 1 control female, one female of MD and 2 HD females, wet nose in one HD female and crust on back in one MD female observed and considered to be incidental in nature.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance. None of the females showed signs of abortion or premature delivery. During the weekly detailed clinical observation, no relevant differences between the groups were found.

Mortality
During the treatment period of this study, one each mortality and morbidity was observed as follows:
- Male No. 25 (MD) was found dead on premating day (PMD) 18. No specific clinical signs were observed in this animal before and on day of death.
- Male No. 34 (HD) was sacrificed in moribund condition due to animal welfare reasons on PMD 15. Predominant clinical signs observed in this animal before and on day of moribund sacrifice were moving the bedding and diarrhoea from PMD 8-15, alopecia on right cheek, marked piloerection and moderate salivation from PMD 14-15 and moderately reduced spontaneous activity on day of moribund sacrifice i.e. PMD 15.
Macroscopically, male No. 25 (MD) observed for lung failure to collapse and male no. 34 (HD) with small thymus at necropsy.
Histopathologically, for both animals, the cause of mortalitiy/morbidity was not evident at histopathology evaluation.


Body Weight Development
In both males and females, there was no test item treatment related effect observed on group mean body weight in the male LD, MD and all female treatment groups during the entire study period when compared with the controls. However lower group mean body weight in HD males was observed throughout the study from premating day 7 when compared to the controls but statistical significance was achieved only for group mean body weight on premating day 7 and 14. In females, no relevant effect on group mean body weight was observed in treatment groups throughout the study period when compared with the controls.
In males, statistically significantly lower group mean body weight gain was observed in HD group during premating day 1-7, 7-14, premating day 1- post mating day 18 and premating day 1- terminal sacrifice when compared to the controls (C1). There was statistically significantly higher group mean body weight observed in HD group males during premating day 14-20. In females, statistically significantly higher group mean body weight gain was observed during premating day 14-20 in HD group when compared with the controls. A slight but attenuated mean daily weight gain in female animals of the MD group was also observed during premating day 14 to 20 and not assumed to be toxicologically relevant as absolute body weights were within the normal range of variation throughout the study period and did not differ significantly from the controls.
As there was no effect observed on overall health of the animals, food consumption, blood parameters, histopathology and group mean values were not more than 10% different from controls, this effect on body weight development in HD males was not considered to be adverse.


Food Consumption
In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group.
No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.


Haematology and Coagulation
In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group. However, statistically significantly higher reticulocytes count in HD was observed when compared with the controls.
In females sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology parameters. However, statistically significantly lower reticulocytes count in MD and HD was observed when compared with the controls.
As all group mean and individual values for reticulocytes in male and females were within historical data range (Male - 1.1- 2.4 %, Female- 0.63 -7.7 %) and there was no effect on RBC values, this effect on male reticulocytes in HD group and female MD and HD group was not considered to be of toxicological relevance.
Besides, all haematological parameters and blood coagulation parameters in male and females were within the normal range of variation and differences are not assumed to be biologically relevant.
No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.


Clinical Biochemistry
In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the respective controls except statistically significantly higher group mean alkaline phosphatase in HD group females were observed when compared with the controls.
As group mean alkaline phosphatase value in female HD group was within historical control data range (AP- 45.14- 789.94 U/L) this effect in female HD group was not considered to be adverse.
Besides, all parameters of clinical chemistry were within the normal range of variation for this strain and differences between dose and control groups are not assumed to be biologically relevant.


Urinanalysis
The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. Slightly high protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Functional Observations
In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.
In females, statistically significantly lower not supported rearing count in LD and HD groups in the last week of treatment were observed when compared to the controls. Due to lack of dose dependency and consistency, it has no toxicological relevance and could be considered as biological variation. There were no biologically relevant differences in body temperature between the groups.


Organ Weights
In males, statistically significantly higher relative (to body weight) liver weights in HD group (17% higher than control: absolute mean 13.75g compared to 12.18g in the control) were observed when compared with the controls. There was also higher relative spleen weight (16 % higher than control), higher absolute and relative thyroid/ parathyroid weight (124 and 131.5 % higher than control) and higher relative kidney weights (15% higher than control) in HD group was observed without achieving statistical significance when compared with the controls.
In females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group. However, higher relative kidney weights in HD group (17% higher than control), lower absolute and relative thyroid/ parathyroid weight in LD, MD and HD group (8.88 - 23.29 % lower than control) and lower absolute and relative uterus with cervix weight in HD group (12.57- 14.90 % lower than control) was observed without achieving statistical significance.
In the light of fact that no test item related histopathological findings and effects on liver enzymes were observed, this effect on few male and female organ weights was not considered to be adverse.


Pathology
Few specific macroscopic changes like lung failure to collapse (MD found dead male no. 25), fluid filled cyst on the right kidney with a diameter of 0.4 cm (MD terminally sacrificed male no. 22) and small thymus (HD found dead male no. 34) were recorded in the male animals, which based on microscopic examination were not considered to be of test item treatment relevance. No macroscopic findings were recorded in any female animal.
The above mentioned findings were deemed incidental and there were no gross lesions in any animal that could be attributed to treatment with the test item.


Histopathology
In decedents (male no. 25 and 34), all histopathological findings recorded were deemed incidental or were within the range of background alterations that may be recorded in Wistar rats.
In terminally sacrificed animals, in the stomach of some animals from the control and high dose group squamous hyperplasia, minimal to slight hyperkeratosis and minimal to slight mixed cell infiltrates were recorded. The observed gastric changes were located mostly at the limiting ridge and were considered most likely incidental. However, a mechanical irritation of the applied bolus or a direct action of the applied test item cannot be fully excluded.
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina.
For Sperm staging evaluations the testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.
In conclusion, under the conditions of this study, there were neither macroscopic lesions nor histological changes that could be clearly attributed to treatment with test item.

Thyroid Hormone (T4) Analysis
No test item related effect of toxicological relevance or statistical significance was observed on male in the treatment groups.
Besides, no test item related histopathological findings were observed in thyroid/parathyroid gland of selected parental animals and there was also no test item related effect observed on T4 hormone level in parental males.






Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen in any of the doses ( 100 // 300 // 1000 mg a.i./kg bw) in the main OECD 422 study.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Sodium oleylamphopolycarboxyglycinate, (1,3 -Propanediamine, N1 -(3 -aminopropyl)-N3 -[3 -[(9Z)-9 -octadecen-1 -ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541 -49 -1) in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
- There was one mortality observed in the study (male no. 25 of MD group) on premating day 18 and one male (male no. 34 of HD group) was sacrificed in moribund condition on premating day 15 due to animal welfare reasons. Histopathologically cause of mortality or morbidity was not evident in both males.
- No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups.
The NOAEL of Sodium oleylamphopolycarboxyglycinate, (1,3 -Propanediamine, N1 -(3 -aminopropyl)-N3 -[3 -[(9Z)-9 -octadecen-1 -ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541 -49 -1) in this study for general toxicity and reproductive toxicity screening is considered to be 1000 mg/kg bw/day.
Executive summary:

The aim of this study was to assess the possible effects of Sodium oleylamphopolycarboxyglycinate, (1,3 -Propanediamine, N1 -(3 -aminopropyl)-N3 -[3 -[(9Z)-9 -octadecen-1 -ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541 -49 -1) on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 69 days, i.e. during 20 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 37-38 days were completed. Animals of the control group were handled identically as the dose groups but received aqua ad iniectabilia (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistarrats. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                 0      mg/kg bw/day

Low Dose:             100   mg/kg bw/day

Medium Dose:       300   mg/kg bw/day

High Dose:            1000 mg/kg bw/day

The test item was dissolved in aqua ad iniectabilia and administered daily during 20 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for 37-38 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males and females from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment from all animals and in the last week of treatment infive randomly selected males and femalesof each group.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum.

The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment day 38-39 and the females along with their pups were sacrificed on post natal day 13.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natalday 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on control , high dose and dead animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in any organ/tissues of the high dose group. For the testes, a detailed qualitative examination was made taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

Mortality:

There was one mortality observed in the study (male no. 25 of MD group)on premating day 18 and one male (male no. 34 of HD group) was sacrificed in moribund condition on premating day 15 due to animal welfare reasons. Histopathologically cause of mortality or morbidity was not evident in both males.

Clinical Observations:

In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were moderate salivation and moving the bedding on one day in 1 LD male and on few days in one MD male. Moving the bedding in all HD males and slight to moderately increased salivation in 2 HD males was also observed on few days during premating day 1 to mating and post mating day 14. Abnormal breathing in 2 HD males was observed between premating day 2-10.

In terminally sacrificed females, major clinical signs observed during the treatment period (premating day 1 to PND 12) were moving the bedding in 3 MD (on few occasions between PMD 17 - PND 12) and in all HD animals (between PMD 1 - PND 12 on majority days). A slight to moderately increased salivation was observed in 2 HD group females (few days between PMD 19 and PND 12). There was also moderate piloerection in one MD female on PND 0, slightly reduced spontaneous activity in one LD female on PMD 6 and in 2 HD females (No. 76 on PMD 1 and No. 71 on PMD 1-5 and 9-10), ataxia in one HD female No. 71 on PMD 1-5 and 9-10, abnormal breathing in one HD female on PND 4 and regurgitation of a part of the test item in one MD and HD female (No. 74 on GD 16 of the HD group, No. 62 on PND 11 of the MD group) observed.

None of the females showed signs of abortion or premature delivery.

During the weekly detailed clinical observation, no relevant differences between the groups were found.

Functional Observations:

In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

In females, statistically significantly lower not supported rearing count in LD and HD groups in the last week of treatment were observed when compared to the controls. Due to lack of dose dependency and consistency, it has no toxicological relevance and could be considered as biological variation. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development:

In both males and females, there was no test item treatment related effect observed on group mean body weight in the male LD, MD and all female treatment groups during the entire study period when compared with the controls. However lower group mean body weight in HD males was observed throughout the study from premating day 7 when compared to the controls but statistical significance was achieved only for group mean body weight on premating day 7 and 14. In females, no relevant effect on group mean body weight was observed in treatment groups throughout the study period when compared with the controls.

In males, statistically significantly lower group mean body weight gain was observed in HD group during premating day 1-7, 7-14, premating day 1- post mating day 18 and premating day 1- terminal sacrifice when compared to the controls (C1). There was statistically significantly higher group mean body weight observed in HD group males during premating day 14-20. In females, statistically significantly higher group mean body weight gain was observed during premating day 14-20 in HD group when compared with the controls. A slight but attenuated mean daily weight gain in female animals of the MD group was also observed during premating day 14 to 20 and not assumed to be toxicologically relevant as absolute body weights were within the normal range of variation throughout the study period and did not differ significantly from the controls.

Food Consumption:

No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Thyroid Hormone (T4) Analysis:

No test item related effect of toxicological relevance or statistical significance was observed on male thyroxine hormone (T4) in the treatment groups.

Haematology and Coagulation:

In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group. However, statistically significantly higher reticulocytes count in HD was observed when compared with the controls..

In females sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology parameters. However, statistically significantly lower reticulocytes count in MD and HD was observed when compared with the controls.

No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.

Clinical Biochemistry:

In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the respective controls except statistically significantly higher group mean alkaline phosphatase in HD group females were observed when compared with the controls.

Urinalysis:

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. Slightly high protein levels were found in the urine of few male and females of all groups including control group.

Pathology:

Few specific macroscopic changes like lung failure to collapse (MD found dead male no. 25), fluid filled cyst on the right kidney with a diameter of 0.4 cm (MD terminally sacrificed male no. 22) and small thymus (HD found dead male no. 34) were recorded in the male animals, which based on microscopic examination were not considered to be of test item treatment relevance. No macroscopic findings were recorded in any female animal.

Organ Weight:

In males, statistically significantly higher relative (to body weight) liver weights in HD group were observed when compared with the controls. There was also higher relative spleen weight, higher absolute and relative thyroid/ parathyroid weight and higher relative kidney weights in HD group was observed without achieving statistical significance when compared with the controls.

In females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group. However, higher relative kidney weights in HD group, lower absolute and relative thyroid/ parathyroid weight in LD, MD and HD group and lower absolute and relative uterus with cervix weight in HD group was observed without achieving statistical significance.


 

Histopathology:

In decedents (male no. 25 and 34), all histopathological findings recorded were deemed incidental or were within the range of background alterations that may be recorded in Wistar rats.

In terminally sacrificed animals, in the stomach of some animals from the control and high dose group squamous hyperplasia, minimal to slight hyperkeratosis and minimal to slight mixed cell infiltrates were recorded. The observed gastric changes were located mostly at the limiting ridge and were considered most likely incidental. However, a mechanical irritation of the applied bolus or a direct action of the applied test item cannot be fully excluded.

There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina.

In conclusion, under the conditions of this study, there were neither macroscopic lesions nor histological changes that could be clearly attributed to treatment with test item.

Dose Formulation Analysis:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period as measured concentrations were within acceptance criterion of 15%.

Conclusion

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Sodium oleylamphopolycarboxyglycinate, (1,3 -Propanediamine, N1 -(3 -aminopropyl)-N3 -[3 -[(9Z)-9 -octadecen-1 -ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541 -49 -1) in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

-   There was one mortality observed in the study (male no. 25 of MD group) on premating day 18 and one male (male no. 34 of HD group) was sacrificed in moribund condition on premating day 15 due to animal welfare reasons. Histopathologically cause of mortality or morbidity was not evident in both males.

-   No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups. 

The NOAEL of Sodium oleylamphopolycarboxyglycinate, (1,3 -Propanediamine, N1 -(3 -aminopropyl)-N3 -[3 -[(9Z)-9 -octadecen-1 -ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541 -49 -1) in this study for general toxicity and reproductive toxicity screening is considered to be 1000 mg/kg bw/day.