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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
54 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

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:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Fatty acids, C16-18-, reaction products with diethanolamine
Product: Cepreton AT liq. Conc.
Specification: Amide Intermediate of cepreton AT (REACH)
CAS No.: 91032-08-5
Batch No.: ITA0058330
Physical State: solid
Colour: yellow
Molecular Weight: ca.610-872
pH: 7-8
Active Components: >99%
Storage Conditions: at room temperature, protected from light
Date of Analysis: 01.02.2012
Expiry Date: 11.01.2013

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System:

Species/strain: 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: males: 10-11 weeks old, females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups: males: 238 - 278 g
(mean: 254.93 g, ± 20% = 203.94 – 305.91 g) females: 158- 192 g (mean: 177.45 g, ± 20% = 141.96 – 212.94 g)

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 (lot no. 1039)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no160812)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test item was weighed into a tarred plastic vial on a precision balance.
The dose formulations were prepared by adding the small volume of sterile water and further vortexing it for 2-3 minutes. The required volume was brought by adding further volume of sterile water. The formulation sample was subjected to homogenizer and then kept in water bath (70oC) for maximum 1 min.
The vehicle was selected based on the test item’s characteristics. The test item formulations were prepared freshly on each administration day before the administration procedure. The homogeneity was guaranteed by intensive stirring during application.

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of maximum 54 days, i.e. during 14 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 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
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
Duration of treatment / exposure:
maximum 54 days, i.e. during 14 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 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000
Basis:
other: As it is
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
Control animals:
yes, concurrent vehicle
Details on study design:
Number and Sex of the Animals:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Preparation of the Animals:

Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals 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.

Experimental Groups and Doses:

According to the results of the dose range finding study and in consultation with the sponsor the following doses are selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
Control: 0 mg/ kg body weight
Low Dose (LD): 100 mg/kg body weight
Medium Dose (MD): 300 mg/kg body weight
High Dose (HD): 1000 mg/kg body weight

Administration of Doses:

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.

Body Weight and Food Consumption:

The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period 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 off parturition (day 0 post-partum) as well as day 4 post-partum along with pups.
Food consumption was measured weekly 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.

Mating:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smears of the females were checked every morning after the start of the mating period to confirm the copulation. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Clinical Observations:

General clinical observations were made once a day. Once before the first exposure, and 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.

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 on lactation days in 5 randomly selected females (only lactating females were evaluated) 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).

Litter Observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. 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 and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Haematology:

Haematological parameters from five randomly selected males and females of each group were examined at the end of the treatment .
Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined .

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).

Blood Coagulation:

Coagulation parameters from five selected males and females of each group were examined at the end of the treatment .
Blood from the abdominal aorta of the animals was collected in citrate-coated tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT)

Clinical Biochemistry:

Parameters of clinical biochemistry from five selected males and females of each group were examined at the end of the treatment .
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), potassium (K).

Urinalysis:

A urinalysis was performed with samples collected from 5 selected males at necropsy. Additionally, urine colour/ appearance were recorded.
The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes.

Pathology:
Gross necropsy-
All male animals were sacrificed after the completion of the mating period (total dosing period: 28/ 29 days) on study day 29/ 30, while female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 3:1, medistar Arzeneimittel, lot no: 00312, expiry date: 06/2014, Serumwerk, lot no: 00312) was used. Pups were killed on PND 4 by decapitation.

Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Females (animals 46, 56, 63, 78 and 80) showing no sign of pregnancy was sacrificed 26 days after the last day of the mating period.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights:

The wet weight of the organs of 5 males and 5 females selected from each group was recorded as soon as possible. Paired organs were weighed separately. In addition reproductive organs of all animals were weighed.
Organs Weighed at Necropsy:
liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart

The following tissues of the same selected animals from each group were preserved in 10% neutral buffered formalin except eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin.
Preserved and Examined Tissues:

brain (cerebrum, cerebellum and pons), ovaries (females), spinal cord, uterus with cervix (females), liver, vagina (females), kidneys, testes (males)
adrenal glands, epididymides (males), stomach, prostate and seminal vesicles with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder, thymus, lymphnodes (mesentric and axillary), Thyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, bone with bone marrow (sternum), lung and trachea, pituitary gland, mammary glands, oesophagus, heart, gross lesions.

Histopathology:

All organs and tissues were evaluated from selected males and females of the control and high dose group:
Males Nos.: 1, 4, 6, 8, 9, 32, 36, 37, 38, 40; Females Nos.: 41, 42, 48, 49, 50, 71, 73, 74, 75, 79.
Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and macroscopic changes were evaluated in all study animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.





Examinations

Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no effects on food intake
Food consumption and compound intake (if feeding study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality:

There was no mortality noted in treated and control groups during the study period.

Clinical Observations:

There were few clinical signs namely nasal discharge, alopecia, piloerection, eschar, moving the bedding and salivation recorded in male or female animals of LD or MD or HD groups, which were isolated and transient. These findings were not considered to be of toxicological relevance.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observations:

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development:

In both males and females, no treatment related changes were noted for body weight and body weight change during the study period. Statistically there was significant decrease noted for mean body weight change in female LD group on 1st week of premating period when compared to the corresponding control. Due to lack of a dose response pattern this change was not related to treatment.

Food Consumption:

In both males and females, no treatment related changes were noted for treated group when compared to corresponding control. There was no statistically significant difference in food intake noted between the treated and control groups.

Litter Data:

No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4. The statistical evaluation of data revealed significant decrease in number of female pups on PND 0 and 4 in MD group. But due to lack of dose response pattern this finding was not related to treatment.

Litter Weight Data:

No treatment related changes were noted for the mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 in treated groups when compared to corresponding control. However, there was statistically significant decrease in female litter weight in MD group on PND 0 and 4. This was evident by lower number of female pups and in addition there was lack of dose response. Hence, this finding was considered to have no toxicological relevance.

Precoital Interval and Duration of Gestation:

No treatment related changes were noted for the precoital interval and duration of gestation in treated groups when compared to control. All pregnancies resulted in normal births.
Successful mating resulted 90% pregnancy rates in C, LD, MD groups and 80% in HD group.

Pre- and Post-Natal Data:

No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre and post implantation loss in treated groups when compared to control. There were no statistically significant differences noted between the treated and control group values.

Reproductive Indices:

There were no treatment related changes noted for copulation index (%), fertility index (%) and delivery index (%) in treated groups when compared to corresponding control group.

Pup Survival Data:

No treatment related changes were noted for survival of the pups from PND 0 to PND 4 in treated group when compared to controls.

Pup External Findings:

No treatment related gross external findings were observed in of the treated groups.

Haematology and Coagulation:

There were no treatment related changes noted for haematological and coagulation parameters in male and female treated groups when compared to the corresponding control. However, there was statistically significant increase in mean platelet, lymphocyte and monocyte values in male MD group when compared to corresponding control. In the absence of a dose response pattern the above findings were considered to have no toxicological relevance.

Clinical Biochemistry:

There was no treatment related changes noted for measured clinical biochemistry parameters of male and female treated groups when compared to corresponding control. There were no statistically significant differences between the treated and control group values.

Urinalysis:

The urinalysis performed in male animals revealed no treatment related effect in treated groups when compared to the control group.

Pathology:

One control female, one female of LD group, one female of MD group and two females of HD group did not show any indication of recent pregnancy at terminal sacrifice. As this was not dose-related, it was not considered to be related to treatment.
At terminal sacrifice, macroscopic organ findings noted were few, and none of them was considered to be test item-related.

Organ Weight:

There were no changes noted for organ weight in both males and females considered to be related to treatment when compared to corresponding control. There were no statistically significant differences noted between the treated and control values except for relative (to body weight) weight of thymus in male MD group. This change in the absence of dose response pattern and lack of histological findings was not considered to be related to treatment.
However, there was slight decrease noted in absolute and relative (to terminal body weight) weight of spleen in male MD group, increase in absolute and relative (to terminal body weight) weight of adrenal gland in male HD group, increase in absolute and relative (to terminal body weight) weight of thyroid gland in male LD group, decrease in absolute weight of thymus in male MD group, increase in absolute weight of liver in female HD group, decrease in relative (to terminal body weight) weight of thyroid gland in female MD group, increase in relative (to terminal body weight) weight of thymus in female LD and HD groups. In the absence of statistical significance and histopathological findings the above changes were not related to treatment.

Histopathology:

Reproductive organs-

No test item-related effects were noted on male and female reproductive organs in this study.
In the males, spermatic granuloma(s) were seen in the epididymis in one control rat and two rats HD group, and were considered to be a spontaneous change, as this is occasionally observed in untreated male rats of this strain and age.
Most females showed histological evidence of preceding pregnancy. The number of large ovarian corpora lutea was essentially similar in all study groups.
One control female (No. 46), one female of LD group (No. 56) one female of MD group (No. 63) and two females of HD group (Nos. 78 and 80) did not show any indication of recent pregnancy at terminal sacrifice. Histomorphology of their reproductive organs indicated physiological sexual cycling.

Other organs-

No test item-related histopathological findings were noted in the other organs and tissues evaluated in randomized males and females of the control and high dose group. Histopathological changes seen at terminal sacrifice were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions.

Effect levels

Dose descriptor:
NOAEL
Remarks:
based on general and reproductiva/developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of the Fatty acids, C16-18-, reaction products with diethanolamine to the male (minimum 28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance in male and female animals. There were also no toxicologically relevant findings noted for reproductive and developmental parameters.
Based on the data generated from this “Combined Repeated Dose Oral Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test with Fatty acids, C16-18-, reaction products with diethanolamine, the no observed adverse effect level (NOAEL) for general and reproductive and developmental toxicity is considered to be 1000 mg/kg body weight.
Executive summary:

The aim of this study was to assess the possible effects of Fatty acids, C16-18-, reaction products with diethanolamine 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 maximally 54 days for females and minimum of 28 days for males. Animals of control group were handled identically as the dose groups but received same dose volume of the vehicle used in this study. Each of the 4 groups comprised 10 male and 10 female Wistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, 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 performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations were performed in the week before the treatment and at the end of the study on five randomly selected males and females.

After 14 days of treatment to both male and female animals were mated (1:1) for a maximum of 14 days. From 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 off parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on GD 26 from the day of confirmed mating.

Pups sacrificed on post natal day 4, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. Reproductive organs were evaluated in all study animals.

The following doses were evaluated: Control: 0 mg/kg body weight; Low Dose: 100 mg/kg body weight; Medium Dose: 300 mg/kg body weight; High Dose: 1000 mg/kg body weight.

The test item formulation was prepared freshly on each day of administration. The test item was suspended in sterile water and administered daily during 14 days of pre-mating and during mating period in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28 days. Non pregnant females were treated until day 25 after confirmed mating. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Summary Results:

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

There were no clinical signs considered to be of toxicological relevance recorded in male and female animals of treated groups.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

In both males and females, no treatment related changes were considered for body weight, body weight change and food consumption in treated groups when compared to control.

No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4.

No treatment related changes were noted for the mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 in treated groups when compared to corresponding control.

No treatment related changes were noted for the precoital interval and duration of gestation in treated groups when compared to control. All pregnancies resulted in normal births.

No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre and post implantation loss in treated groups when compared to control.

There were no treatment related changes noted for copulation index (%), fertility index (%) and delivery index (%) in treated groups when compared to corresponding control group.

No treatment related changes were noted for survival of the pups from PND 0 to PND 4 in treated group when compared to control.

No treatment related changes were noted for viability index (%). No treatment-related gross external findings were observed in the treated groups.

There were no statistically significant changes considered to be of toxicological relevance noted for haematological, clinical biochemistry and coagulation parameters in male and female treated groups when compared to the corresponding control.

The urinalysis performed in male animals revealed no test item related effect in the treated groups when compared to control.

One control female, one female of LD group, one female of MD group and two females of HD group did not show any indication of recent pregnancy at terminal sacrifice. As this was not dose-related, it was not considered to be related to treatment.

At terminal sacrifice, macroscopic organ findings noted were few, and none of them was considered to be test item-related.

There were no changes noted for organ weight in both males and females considered to be related to treatment when compared to corresponding control.

No test item-related histopathological findings were noted in the male and female reproductive organs and other organs and tissues evaluated in males and females of the control and high dose group.