Fatty acids, C16-18, reaction products with diethanolamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

After subacute treatment no effects on fertility parameters were detected in any animal.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Route of administration:

oral: gavage

Vehicle:

water

Details on 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 (70°C) 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.

Details on mating procedure:

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.

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

Details on study schedule:

Arrival of the Test Item: 11 June 2012
Study Initiation Date: 15 November 2012
Experimental Starting Date: 26 November 2012
Experimental Completion Date: 22 January 2013
Study Completion Date: May 2013

Remarks:

Doses / Concentrations:
100, 300, 1000
Basis:
nominal conc.

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 selected males and on lactation days in 5 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 (minimum dosing period: 28 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.

Parental animals: Observations and examinations:

Mortality, Clinical Observations, Functional Observations, Body Weight Development, Food Consumption, Haematology and Coagulation, Clinical Biochemistry, Urinalysis, Pathology, Organ Weight, Histopathology.

Litter observations:

Litter Data, Litter Weight Data, Precoital Interval and Duration of Gestation, Pre- and Post-Natal Data, Reproductive Indices, Pup Survival Data, Pup External Findings.

Postmortem examinations (parental animals):

Haematology and Coagulation,Clinical Biochemistry, Urinalysis, Pathology, Organ Weight, Histopathology.

Postmortem examinations (offspring):

Gross external examination at necropsy

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).

Reproductive indices:

copulation index (%), fertility index (%) and delivery index (%)

Offspring viability indices:

viability index (%)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

After subacute treatment no effects on fertility parameters were detecetd in male and female animals. Numbers of live pups were in the expected range and comparable to concurrent controls.

Effects on developmental toxicity

Description of key information

No effects on developmental parameters were detected in any animal up to the limit dose of 1000 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Age at arrival: 9 weeks (females), 11 weeks (males)
- Weight at arrival: 320-339 g (males), 190-214 g (females)
- Fasting period before study: no
- Housing: during pre-pairing phase and after mating: max. 5 of one sex to a cage, in polisulphone solid bottomed cages; during mating: 1 m + 1 f in clear polisulphone cages with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l.) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% carboxymethyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations of the test item were prepared as suspensions in 0.5% CMC. Concentration and homogeneity of the low and high dose level were assessed by taking six analytical aliquots in different positions. For the intermediate levels, only concentration was assessed by taking two different analytical aliquots.
28 hour stability at room temperature and a 8 day stability at +4°C were verified in the range from 10 to 100 mg/mL.
The results of the analyses were within the acceptability limits (85-115% for concentration and CV < 10% for homogeneity).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: ???
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 through day 19 post coitum

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on information from previous study (reproduction/developmental toxicity screening test)
- Rationale for animal assignment (if not random): computerised stratified randomisation to give approximately equal initial group mean body weights

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 9, 12, 15, and 20 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
6, 9, 12, 15 and 20 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: abnormalities

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placentae

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicological significance were noted during the study and no signs of reactions to treatment were observed during the dosing period. The scab on the neck seen in one high dose female during the last day of study was considered of no relevance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences were noted in body weight and body weight gain of females during the study, between control and treated groups. No importance was attributed to the slight statistically significant increase in body weight gain on gestation Day 9 in group 4 females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No relevant changes were detected in food consumption between treated and control females

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicological relevant changes were seen in uterus weight in treated groups compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic changes were observed at post mortem examination in treated animals, when compared to the controls. The only macroscopic change, consisting of scab on dorsal neck, is suggested to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Four females in the low dose group and one each in the mid- and high dose groups were found not pregnant at necropsy. Since the mating occurred
before the start of treatment, the incidence of non pregnant females is unrelated to treatment.
One female in each control, low and mid-dose groups had unilateral implantation. The number of females with live foetuses on gestation Day 20 was 24 in the control group, 20 in the low dose and 23 in the mid- and high dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: no adverse effects observed up to the limit dose

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

A total of 5 small foetuses (< 2.7 g) were detected; 1 out of 317 in the control, 2 out of 280 in the low dose, 1 out of 304 in the mid-dose group and 1 out of 322 in the high dose group.
Since the number of litters affected was similar in the control and treated groups, the finding was considered incidental. No other abnormalities were detected at the external examination of foetuses.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

The skeletal findings did not give rise to concern. Anomalies (AN) and variations (VA) were observed, but they were distributed in a similar way in dose groups and control. Therefore they were considered not treatment related. The only malformation (Forepaw Flexure) was limited to control (one fouetus) and low dose group (3 foetuses/2 litters) and, hence, not dose related. Due to the low incidence and the presence only in the low dose group, this finding was considered incidental.

Visceral malformations:

no effects observed

Description (incidence and severity):

The visceral examination does not give rise to concern. Anomalies (AN) and variations (VA) were observed, but their incidence was similar in the treated groups and the control, and in some cases even more pronounced in the control.
Extreme pelvic dilatation (malformation) was seen in three foetuses/three litters in controls and in one foetus each in the low and high dose group. Due to the lower incidence observed in treated groups respect to controls, this change was considered not related to treatment.
Ureters extremely enlarged and/or kinked were seen in treated and control groups, with similar or lower incidence respect to controls and therefore this finding was considered not treatment-related.
A pulmonary cyst was reported for one foetus in the mid-dose group. Due to the lowincidence and lack of dose-response relationship, this change was considered not related to treatment.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the limit dose

Abnormalities:

no effects observed

Developmental effects observed:

no

FATE OF FEMALES

	0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Initial group size	24	24	24	24
Not pregnant	0	4	1	1
Unilateral implantation	1	1	1	0
With live foetuses at gestation Day 20	24	20	23	23

 

BODY WEIGHT (g) OF FEMALES WITH LIVE FOETUSES ON GESTATION DAY 20 – GROUP MEAN DATA

Dose level [mg/kg bw/d]		days
		0	6	9	12	15	20
0	(n) Mean SD	24 234.86 8.55	24 264.87 8.56	24 274.20 10.33	24 288.52 9.93	24 306.45 12.43	24 379.71 21.44
10	(n) Mean SD	20 235.79 8.03	20 265.86 8.59	20 274.87 9.07	20 289.63 9.71	20 307.29 12.52	20 381.31 27.30
300	(n) Mean SD	23 235.98 11.70	23 266.94 12.76	23 277.94 12.98	23 291.47 13.02	23 310.26 15.29	23 383.07 23.35
1000	(n) Mean SD	23 236.99 10.17	23 263.97 8.70	23 276.26 9.53	23 290.95 10.77	23 311.93 10.59	23 389.81 16.25

 

BODY WEIGHT GAIN PER DAY (g) OF FEMALES WITH LIVE FOETUSES ON GESTATION DAY 20 – GROUP MEAN DATA (mean daily body weight gain over the previous period starting from gestation day 0)

Dose level [mg/kg bw/d]		days
		6	9	12	15	20
0	(n) Mean SD	24 5.001 0.771	24 3.112 1.349	24 4.772 1.203	24 5.977 2.077	24 14.652 2.805
10	(n) Mean SD	20 5.013 0.654	20 3.002 1.548	20 4.920 1.519	20 5.887 2.021	20 14.804 3.328
300	(n) Mean SD	23 5.159 1.125	23 3.668 1.036	23 4.508 1.350	23 6.266 1.505	23 14.561 2.275
1000	(n) Mean SD	23 4.497 1.237	23 4.098* 1.379	23 4.895 1.469	23 6.994 1.670	23 15.576 1.796

 

* = mean value of group is significantly different from control at p < 0.05

LITTER DATA AND SEX RATIOS - GROUP MEAN DATA

Dose level [mg/kg bw/d]		Corpora lutea	Implan-tations	Uterine deaths			Viable young			% males	Implantation loss [%]			Litter weight [g]	Mean foetal weight [g]
				Early	Late	Total	Total	M	F		Pre	Post	Total
0	Mean SD (n)	13.88 3.04 24	13.83 3.03 24	0.63 1.01 24	0.00 0.00 24	0.63 1.01 24	13.21 3.20 24	6.65 2.35 23	6.83 2.01 24	47.72 14.08 23	0.28 1.37 24	5.44 9.24 24	5.71 9.16 24	47.25 12.06 24	3.58 0.23 24
10	Mean SD (n)	14.40 3.70 20	14.20 3.99 20	0.20 0.70 20	0.00 0.00 20	0.20 0.70 20	14.0 4.14 20	6.63 2.27. 19	8.11 1.70 19	45.89 9.49 18	3.06 8.73 20	3.44 11.74 20	5.66 15.69 20	49.62 14.38 20	3.60 0.29 20
300	Mean SD (n)	13.61 2.50 23	13.57 2.52 23	0.35 0.57 23	0.00 0.00 23	0.35 0.57 23	13.22 2.33 23	6.65 2.69 23	6.57 2.45 23	50.07 16.57 23	0.33 1.61 23	2.33 3.82 23	2.64 4.54 23	47.26 8.19 23	3.59 0.21 23
1000	Mean SD (n)	14.74 2.03 23	14.61 1.90 23	0.57 0.79 23	0.04 0.21 23	0.61 0.84 23	14.00 2.07 23	7.04 2.02 23	6.96 2.23 23	50.35 14.50 23	0.78 2.06 23	4.23 6.01 23	4.96 6.35 23	52.02 7.03 23	3.73 0.25 23

 

EXTERNAL EXAMINATION OF FOETUSES – GROUP INCIDENCE

Dose level [mg/kg bw/d]	Organ	Cat	Observation	No. foetuses			No. litters
				Observed	Affected	%	Observed	Affected	%
0	Whole foetus		No abnormalities detected	317	316	99.68	24	-	-
	Whole foetus	AN	small	317	1	0.32	24	1	4.17
10	Whole foetus		No abnormalities detected	280	278	99.29	20	-	-
	Whole foetus	AN	small	280	2	0.71	20	2	10.00
300	Whole foetus		No abnormalities detected	304	303	99.67	23	-	-
	Whole foetus	AN	small	304	1	0.33	23	1	4.35
1000	Whole foetus		No abnormalities detected	322	321	99.69	23	-	-
	Whole foetus	AN	small	322	1	0.31	23	1	4.35

 

SKELETAL EXAMINATION OF FOETUSES – GROUP INCIDENCE

Dose level [mg/kg bw/d]	Organ	Cat	Observation	No. foetuses			No. litters
				Observed	Affected	%	Observed	Affected	%
0	Forepaw(s)	AN	Metacarpal(s) no ossification 4^th	164	62	37.80	24	17	70.83
	Forepaw(s)	MA	Flexure	164	1	0.61	24	1	4.17
	Hindpaw(s)	AN	Metatarsal(s) no ossification 4^th	164	1	0.61	24	1	4.17
	Hindpaw(s)	MA	Metatarsal(s) incomplete ossification 4th	164	4	2.44	24	4	16.67
	Lumbar vertebrae	AN	Centrum dumb-bell shaped	164	1	0.61	24	1	4.17
	Lumbar vertebrae	AN	Asymmetrical dumb-bell shaped	164	1	0.61	24	1	4.17
	Pelvic girdle	AN	Pubis incomplete ossification	164	4	2.44	24	3	12.50
	Pelvic girdle	AN	Ischium incomplete ossification	164	2	1.22	24	2	8.33
	Ribs	VA	rudimentary 14th	164	32	19.51	24	16	66.67
	Ribs	VA	short 14th	164	1	0.61	24	1	4.17
	Sacral vertebrae	AN	Arch(es) incomplete ossification	164	2	1.22	24	2	8.33
	Skull	AN	Temporal incomplete ossification	164	27	16.46	24	18	75.00
	Skull	VA	Interparietal incomplete ossification	164	1	0.61	24	1	4.17
	Skull	VA	Supraoccipital incomplete ossification	164	5	3.05	24	5	20.83
	Sternebrae	AN	Asymmetrical ossification	164	6	3.66	24	6	25.00
	Sternebrae	AN	Asymmetrical ossification 5th	164	8	4.88	24	5	20.83
	Sternebrae	AN	No ossification 6th	164	6	3.66	24	4	16.67
	Sternebrae	AN	Dumb-bell shaped 5th	164	1	0.61	24	1	4.17
	Sternebrae	VA	Incomplete ossification 5th	164	41	25.00	24	16	66.67
	Sternebrae	VA	Incomplete ossification 6th	164	56	34.15	24	13	54.17
	Sternebrae	VA	No ossification 5th	164	9	5.49	24	6	25.00
	Sternebrae	VA	Incomplete ossification	164	20	12.20	24	10	41.67
	Thoracic vertebrae	AN	Centrum no ossification	164	2	1.22	24	2	8.33
	Thoracic vertebrae	AN	Centrum bipartite	164	1	0.61	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification	164	20	12.20	24	13	54.17
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	164	11	6.71	24	8	33.33
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	164	4	2.44	24	4	16.67
100	Forepaw(s)	AN	Metacarpal(s) no ossification 4^th	145	53	36.55	20	13	65.00
	Forepaw(s)	MA	Flexure	145	3	2.07	20	2	10.00
	Lumbar vertebrae	AN	Centrum dumb-bell shaped	145	1	0.69	20	1	5.00
	Ribs	VA	14 ribs	145	1	0.69	20	1	5.00
	Ribs	VA	rudimentary 14th	145	21	14.48	20	14	70.00
	Ribs	VA	short 14th	145	1	0.69	20	1	5.00
	Skull	AN	Temporal incomplete ossification	145	25	17.24	20	15	75.00
	Skull	VA	Supraoccipital incomplete ossification	145	2	1.38	20	2	10.0
	Skull	VA	Interparietal incomplete ossification	145	1	0.69	20	1	5.00
	Sternebrae	AN	No ossification 6th	145	2	1.38	20	2	10.0
	Sternebrae	AN	Asymmetrical ossification	145	4	2.76	20	3	15.00
	Sternebrae	AN	Asymmetrical ossification 5th	145	5	3.45	20	4	20.00
	Sternebrae	VA	Incomplete ossification 5th	145	35	24.14	20	15	75.00
	Sternebrae	VA	No ossification 5th	145	4	2.76	20	4	20.00
	Sternebrae	VA	Incomplete ossification	145	13	8.97	20	11	55.00
	Sternebrae	VA	Incomplete ossification 6th	145	52	35.86	20	15	75.00
	Thoracic vertebrae	AN	Centrum bipartite	145	1	0.69	20	1	5.00
	Thoracic vertebrae	AN	Centrum no ossification	145	1	0.69	20	1	5.00
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	145	4	2.76	20	4	20.00
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	145	27	18.62	20	16	80.00
	Thoracic vertebrae	VA	Centrum incomplete ossification	145	12	8.28	20	7	35.00
300	Forepaw(s)	AN	Metacarpal(s) no ossification 4^th	150	44	29.33	23	15	65.22
	Lumbar vertebrae	VA	Centrum incomplete ossification	150	2	1.33	23	28.70
	Ribs	AN	Wavy	150	1	0.67	23	1	4.35
	Ribs	VA	rudimentary 14th	150	21	14.00	23	11	47.83
	Skull	AN	Temporal incomplete ossification	150	31	20.67	23	13	56.52
	Skull	VA	Supraoccipital incomplete ossification	150	1	0.67	23	1	4.35
	Skull	VA	Interparietal incomplete ossification	150	6	4.00	23	4	17.39
	Sternebrae	AN	Asymmetrical ossification 5th	150	12	8.00	23	8	34.78
	Sternebrae	AN	No ossification 6th	150	1	0.67	23	1	4.35
	Sternebrae	AN	Asymmetrical ossification	150	8	5.33	23	7	30.43
	Sternebrae	VA	Incomplete ossification	150	9	6.00	23	5	21.74
	Sternebrae	VA	No ossification 5th	150	3	2.00	23	3	13.04
	Sternebrae	VA	Incomplete ossification 6th	150	36	24.00	23	17	73.91
	Sternebrae	VA	Incomplete ossification 5th	150	25	16.67	23	13	56.52
	Thoracic vertebrae	AN	Centrum bipartite	150	1	0.67	23	1	4.35
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	8	5.33	23	6	26.09
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	150	1	0.67	23	1	4.35
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	150	27	18.00	23	15	65.22
1000	Forepaw(s)	AN	Metacarpal(s) no ossification 4^th	164	52	31.71	23	15	65.22
	Hindpaw(s)	VA	Metatarsal(s) incomplete ossification 4th	164	5	3.05	23	3	13.04
	Pelvic girdle	AN	Pubis incomplete ossification	164	1	0.61	23	1	4.35
	Pelvic girdle	AN	Ischiumincomplete ossification	164	1	0.61	23	1	4.35
	Ribs	VA	Rudimentary 14th	164	20	12.20	23	11	47.83
	Ribs	VA	14 ribs	164	2	1.22	23	1	4.35
	Ribs	VA	Short 14^th	164	2	1.22	23	2	8.70
	Skull	AN	Temporal incomplete ossification	164	32	19.51	23	16	69.57
	Skull	VA	Parietalincomplete ossification	164	2	1.22	23	1	4.35
	Skull	VA	Supraoccipital incomplete ossification	164	4	2.44	23	3	13.04
	Skull	VA	Interparietal incomplete ossification	164	4	2.44	23	3	13.04
	Sternebrae	AN	Asymmetrical ossification	164	3	1.83	23	2	8.70
	Sternebrae	AN	Asymmetrical ossification 5th	164	8	4.88	23	6	26.09
	Sternebrae	AN	No ossification	164	1	0.61	23	1	4.35
	Sternebrae	VA	Incomplete ossification 5th	164	30	18.29	23	14	60.87
	Sternebrae	VA	Incomplete ossification	164	8	4.88	23	5	21.74
	Sternebrae	VA	Incomplete ossification 6th	164	33	20.12	23	13	56.52
	Sternebrae	VA	No ossification 5th	164	2	1.22	23	2	8.70
	Thoracic vertebrae	AN	Centrum no ossification	164	1	0.61	23	1	4.35
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	164	14	8.54	23	11	47.83
	Thoracic vertebrae	VA	Centrum incomplete ossification	164	11	6.71	23	8	34.78
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	164	6	3.66	23	6	26.09

 

VISCERAL EXAMINATION OF FOETUSES – GROUP INCIDENCE

Dose level [mg/kg bw/d]	Organ	Cat	Observation	No. foetuses			No. litters
				Observed	Affected	%	Observed	Affected	%
0	Abdomen	VA	Haemorrhagic	153	23	15.03	24	8	33.33
	Great vessels	AN	Innominate artery absent	153	1	0.65	24	1	4.17
	Great vessels	VA	Innominate artery short	153	7	4.58	24	5	20.83
	Heart	AN	Pericardial haemorrhage	153	23	15.03	24	11	45.83
	Heart	AN	Pericardial fluid	153	10	6.54	24	6	25.00
	Heart	AN	Atrium enlarged / slight	153	2	1.31	24	2	8.33
	Heart	VA	Ventricle enlarged / slight	153	8	5.23	24	6	25.00
	Kidneys	AN	Ectopic	153	16	10.46	24	10	41.67
	Kidneys	AN	Pelvic dilatation / moderate	153	5	3.27	24	5	20.83
	Kidneys	AN	Haemorrhagic	153	1	0.65	24	1	4.17
	Kidneys	MA	Pelvic dilatation /extreme	153	3	1.96	24	3	12.50
	Kidneys	VA	Pelvic dilatation / slight	153	6	3.92	24	5	20.83
	Liver	AN	Mottled	153	6	3.92	24	2	8.33
	Testis	AN	Displaced	153	5	3.27	24	4	16.67
	Thoracic cavity	AN	Haemorrhage	153	4	2.61	24	3	12.50
	Ureter	AN	Enlarged/ moderate	153	9	5.88	24	8	33.33
	Ureter	AN	Kinked/ moderate	153	1	0.65	24	1	4.17
	Ureter	MA	Kinked/extreme	153	2	1.31	24	2	8.33
	Ureter	MA	Enlarged/ extreme	153	4	2.61	24	4	16.67
	Ureter	VA	Enlarged/ slight	153	19	12.42	24	12	50.00
	Ureter	VA	Kinked/ slight	153	3	1.96	24	2	8.33
100	Abdomen	VA	Haemorrhagic	135	8	5.93	19	4	21.05
	Great vessels	VA	Innominate artery short	135	1	0.74	19	1	5.26
	Great vessels	VA	Innominate artery longer	135	1	0.74	19	1	5.26
	Heart	AN	Pericardial fluid	135	8	5.93	19	3	15.79
	Heart	AN	Pericardial haemorrhage	135	2	1.48	19	1	5.26
	Heart	VA	Ventricle enlarged / slight	135	3	2.22	19	3	15.79
	Kidneys	AN	Ectopic	135	8	5.93	19	7	36.84
	Kidneys	AN	Pelvic dilatation / moderate	135	1	0.74	19	1	5.26
	Kidneys	MA	Pelvic dilatation /extreme	135	1	0.74	19	1	5.26
	Kidneys	VA	Pelvic dilatation / slight	135	5	3.70	19	5	26.32
	Testis	AN	Displaced	135	7	5.19	19	6	31.58
	Thoracic cavity	AN	Haemorrhage	135	1	0.74	19	1	5.26
	Ureter	AN	Enlarged/ moderate	135	5	3.70	19	4	21.05
	Ureter	AN	Kinked/ moderate	135	1	0.74	19	1	5.26
	Ureter	MA	Enlarged/ extreme	135	2	1.48	19	2	10.53
	Ureter	VA	Enlarged/ slight	135	23	17.04	19	11	57.89
	Ureter	VA	Kinked/ slight	135	6	4.44	19	5	26.32
300	Abdomen	VA	Haemorrhagic	147	25	17.01	23	9	39.13
	Great vessels	VA	Innominate artery longer	147	1	0.68	23	1	4.35
	Heart	AN	Pericardial haemorrhage	147	16	10.88	23	7	30.43
	Heart	AN	Atrium enlarged / slight	147	1	0.68	23	1	4.35
	Heart	VA	Pericardial fluid	147	15	10.20	23	9	39.13
	Heart	VA	Ventricle enlarged / slight	147	7	4.76	23	7	30.43
	Kidneys	AN	Pelvic dilatation / moderate	147	4	2.72	23	4	17.39
	Kidneys	AN	Ectopic	147	13	8.84	23	9	39.13
	Kidneys	VA	Pelvic dilatation / slight	147	5	3.40	23	4	17.39
	Lungs	AN	Lobe abnormal shape	147	1	0.68	23	1	4.35
	Lungs	MA	Pulmonary cyst	147	1	0.68	23	1	4.35
	Testis	AN	Displaced	147	6	4.08	23	5	21.74
	Ureter	AN	Enlarged/ moderate	147	3	2.04	23	2	8.70
	Ureter	MA	Enlarged/ extreme	147	2	1.36	23	2	8.70
	Ureter	MA	Kinked / extreme	147	2	1.36	23	2	8.70
	Ureter	VA	Kinked/ slight	147	7	4.76	23	6	26.09
	Ureter	VA	Enlarged/ slight	147	16	10.88	23	9	39.13
1000	Abdomen	VA	Haemorrhagic	158	10	6.33	23	7	30.43
	Great vessels	VA	Innominate artery short	158	4	2.53	23	3	13.04
	Great vessels	VA	Innominate artery longer	158	1	0.63	23	1	4.35
	Heart	AN	Atrium enlarged / slight	158	1	0.63	23	1	4.35
	Heart	AN	Pericardial haemorrhage	158	22	13.92	23	11	47.83
	Heart	AN	Pericardial fluid	158	7	4.43	23	4	17.39
	Heart	VA	Ventricle enlarged / slight	158	3	1.90	23	2	8.70
	Kidneys	AN	Haemorrhagic	158	1	0.63	23	1	4.35
	Kidneys	AN	Ectopic	158	13	8.23	23	10	43.48
	Kidneys	AN	Pelvic dilatation / moderate	158	2	1.27	23	2	8.70
	Kidneys	MA	Pelvic dilatation / extreme	158	1	0.63	23	1	4.35
	Testis	AN	Displaced	158	2	1.27	23	1	4.35
	Ureter	AN	Kinked/ moderate	158	1	0.63	23	1	4.35
	Ureter	AN	Enlarged/ moderate	158	11	6.96	23	6	26.09
	Ureter	MA	Kinked / extreme	158	2	1.27	23	2	8.70
	Ureter	MA	Enlarged/ extreme	158	1	0.63	23	1	4.35
	Ureter	VA	Enlarged/ slight	158	13	8.23	23	9	39.13
	Ureter	VA	Kinked/ slight	158	7	4.43	23	4	17.39

AN = anomaly

VA = variant

MA = malformation

Conclusions:

Neither clinical signs nor signs of reaction to treatment were noted in treated females. No significant differences were noted in body weight, food consumption, gravid uterus weight, litter data and macroscopic observation of treated females when compared to controls. No changes were noted at the skeletal and visceral examinations of the foetuses which were considered treatment-related.
On the basis of these results the dosage of 1000 mg/kg/day could be considered the NOAEL in this study.

Executive summary:

In a prenatal developmental toxicity study according to OECD Guideline 414 (2001) Fatty acids, C16-18, reaction products with diethanolamine (98.81% a.i.) was administered to 24 female Sprague-Dawley rats in 0.5% carboxymethyl cellulose by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day from days 6 through 19 of gestation.

Body weight, daily clinical signs and food consumption were recorded during thein vivophase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No animals died during the study. Four females in the low dose group and one each in the mid- and high dose groups were found not pregnant at necropsy. One female in control, low and mid-dose groups had unilateral implantation. The number of females with live fetuses on gestation Day 20 was 24 in the control group, 20 in the low dose and 23 in the mid- and high dose groups.

No signs of toxicological significance were noted during the study and no signs of reactions to treatment were observed during the dosing period.

No relevant differences were noted in body weight and body weight gain of females during the study, between control and treated groups.

No relevant changes were detected in food consumption between treated and control females.

No relevant changes were seen in terminal body weight, uterus weight and absolute weight gain, in treated animals compared to controls.

Litter data and sex ratios were not affected by treatment.

No macroscopic changes were observed at post mortem examination in treated animals, when compared to the controls.

A total of 5 small foetuses (<2.7 g) were detected: 1 in each control, mid- and high dose groups and 2 in the low dose group. No other abnormalities were detected at the external examination of foetuses. No changes were noted at the skeletal examination of the foetuses which were considered treatment-related. No changes were noted at the visceral examination of the foetuses which were considered treatment-related.

On the basis of these results the dosage of 1000 mg/kg/day could be considered the NOAEL in this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable guideline study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Neither clinical signs nor signs of reaction to treatment were noted in treated females. No significant differences were noted in body weight, food consumption, gravid uterus weight, litter data and macroscopic observation of treated females when compared to controls. No treatment-related changes were noted at the external, skeletal and visceral examinations of the foetuses. On the basis of these results the dose of 1000 mg/kg bw/day is considered the NOAEL in this study.

Justification for classification or non-classification

Not classified

The available information does not indicate any hazard for reproduction and development.