Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy.
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 176 to 200 g for males and 151 to 175 g for females
- Allocation: The rats were allocated to groups by computerised stratified randomisation to give approximately equal initial group mean body weights
- Housing: 5 of one sex to a cage, in clear polisulphone solid bottomed cages measuring 59.5⇥38⇥20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55 % ± 15 %
- Air changes (per hr): 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Details on mating procedure:
During the mating period, animals were housed on the basis of one male to one female in polysulphone cages measuring 42.5⇥26.6⇥18.5 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. The males were re-caged after mating as they were before mating. After mating, the females were transferred to individual polysulphone solid bottomed cages measuring approximately 42.5⇥26.6⇥18.5 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese) for the gestation period, birth and lactation. Nesting material was provided into suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least twice a week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in the present study in the range from 20 to 200 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspension (r > 0.98; accuracy 90-110%; precision CV < 5%).
Samples of the formulations prepared on Day 1 and Last Week were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (85-115%).
Duration of treatment / exposure:
Males: 5 weeks; females: 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, gestation and post partum periods up to Day 3 post partum.
Frequency of treatment:
Daily
Details on study schedule:
Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray). Each cage tray was checked each morning for the presence of copulation plugs. The female was paired with the same male until positive identification of copulation occurred or 14 days had elapsed.
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Each group comprised 10 male and 10 female rats
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
MORTALITY: all animals were checked early in each working day and again in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Once before com- mencement of treatment (data not tabulated) and daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

NEUROBEHAVIOURAL EXAMINATION:
- FUNCTIONAL OBSERVATION BATTERY TESTS: Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena.
- GRIP STRENGTH AND SENSORY REACTIVITY TO STIMULI: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected (computer generated random order) from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. For males the tests were performed on Day 30 of study and for females on Day 3 post partum.
- MOTOR ACTIVITY ASSESSMENT: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (60 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males the test was performed on Day 30 of study and for females on Day 3 post partum.

BODY WEIGHT: Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination. Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION: The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

CLINICAL PATHOLOGY INVESTIGATIONS: As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation. At the same time interval, individual overnight urine samples were also collected from the same male animals under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. After collection of the urine samples, the water bottles were supplied again to the animals.

HAEMATOLOGY: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count, Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelets
COAGULATION TEST: Prothrombin time

CLINICAL CHEMISTRY: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Inorganic phosphorus, Total bilirubin, Total cholesterol, Bile acids, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: Appearance, Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components
Oestrous cyclicity (parental animals):
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commence- ment of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
Sperm parameters (parental animals):
The testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
1. Tissues specified in section 4.5.6 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term;
2. All abnormalities in all groups.
A detailed qualitative examination of the testes was performed on 5 randomly selected animals in control and high dose groups respectively. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952. The PAS-stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.
Litter observations:
As soon as possible after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
Postmortem examinations (parental animals):
Terminal studies:
- Euthanasia: Parental animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.
Parental Males: The males were killed after the mating of all females, starting from 34 up to 37 days of treatment.
Parental Females: The females with live pups were killed on Day 4 post partum. Three females which did not give birth 25 days after positive identification of mating were killed shortly after (Day 26/27 post coitum). Two females showing no evidence of copulation were killed after 25 days of the last day of the mating session.

- Necropsy: The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
Females
All females were examined also for the following:
– number of visible implantation sites (pregnant animals)
– number of corpora lutea (pregnant animals)
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
Postmortem examinations (offspring):
Terminal studies:
- Euthanasia: Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
- Necropsy: All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. 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. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
Males:
- Copulation Index (%)= (no. of animals mated/no. of animals paired) x 100
- Fertility Index (%)= (no. of males which induced pregnancy/no. of animals paired) x 100

Females:
- Copulation Index (%)= (no. of animals mated/no. of animals paired) x 100
- Fertility Index (%)= (no. of pregnant females/no. of females paired) x 100

Males and females:
Pre coital Interval = Mean number of days between pairing and mating
Offspring viability indices:
- Pre-birth loss was calculated as a percentage from the formula:
(no. of visible implantations total litter size at birth/no. of visible implantations) x 100
- Pre-implantation loss was calculated as a percentage from the formula:
(no. of corpora lutea - no. of visible implantations/no. of corpora lutea) x 100
- Pup loss at birth was calculated as a percentage from the formula:
(total litter size - live litter size/total liter size) x 100
- Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(total litter size at birth - live litter size at Day 4/Total litter size at birth) x 100
- Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.
Clinical signs:
no effects observed
Description (incidence and severity):
No mortality occurred throughout the study and no treatment-related signs were noted.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment in both sexes during the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment in both sexes during the study.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
HAEMATOLOGY: Lymphocytosis was recorded in a number of males dosed with 300 and 1000 mg/kg/day. Lymphocytes mean group values were 36% and 41%, respectively, above controls. The other statistically significant differences between control and treated animals (mean corpuscular haemoglobin concentration in males dosed with 100 mg/kg/day, erythrocytes in females of the same group and basophils in females dosed with 1000 mg/kg/day) were of minimal severity and/or not dose-related, therefore considered of no toxicological significance.
Coagulation: No changes were recorded in the coagulation parameters.

CLINICAL CHEMISTRY: Statistically significant differences between control and treated animals were observed, such as: decrease of triglycerides and chloride in males dosed with 300 mg/kg/day, decrease of albumin in those dosed with 100 mg/kg/day, increase of alkaline phosphatase and decrease of total protein in females treated with 100 mg/kg/day, decrease of globulin in those receiving 100 and 1000 mg/kg/day, increase of albumin/globulin ratio and decrease of creatinine in those treated with 1000 mg/kg/day. Due to the direction of changes and/or the absence of dose-relation, these were considered incidental.

URINALYSIS: Reduced diuresis was recorded in males dosed with 100 mg/kg/day. In addition, urinary haemoglobin, associated with the presence of erythrocytes in the urinary sediment, was recorded in one control animal and one male dosed with 100 mg/kg/day. Due to the absence of dose-relation, the above changes were considered unrelated to treatment.

NEUROBEHAVIOUR:
- Clinical observations (Functional Observation Battery Tests): Neurotoxicity assessment (removal of animals from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test
item.
- Motor activity, grip strength and sensory reaction to stimuli: Motor activity and sensory reaction to stimuli measurements recorded at the end of treatment were comparable between control and treated groups in animals of both sexes. Variations recorded in mean grip strength in Group 4 males receiving 1000 mg/kg bw/day and mean landing foot splay in males receiving 300 and 1000 mg/kg bw/day were considered incidental, since they were observed in one sex only and without correlation with the dose.

HISTOPATHOLOGY: The histopathological changes reported in control and treated animals, such as mucosal erosion in the stomach, lymphoid depletion in the thymus, lymphoid hyperplasia in the spleen or malignant nephroblastoma, seen in a control male, were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.

SPERMATOGENIC CYCLE: A detailed qualitative examination of the testes was performed in five control and high dose group males. Seminiferous tubules were evaluated with respect to their stage in the sperma- togenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

REPRODUCTIVE FUNCTION: Oestrous cycle, reproductive parameters, pairing combination and mating performance
No treatment-related anomalies were noted in the oestrous cycle of the treated females when compared to controls. The mean pre-coital interval and the number of copulation plugs were similar between control and treated groups. All females mated with the exception of one female of the control group. One female of the control group showed unilateral implantation with total resorption. However, 1 female in the control group, 1 in the mid-dose group and 1 in the high dose group were found not pregnant. The copulatory and fertility indices were comparable between control and treated groups.

REPRODUCTIVE FUNCTION: Implantation, pre-birth loss data and gestation length of females
Gestation periods were similar in control and treated groups. In each group, all dams gave birth from Day 21 to 23 post coitum. Corpora lutea, implantations, pre-implantation loss data and total litter size were similar in control and treated groups. The pre-birth loss was higher in females receiving 1000 mg/kg bw/day when compared to the control group. However, this change was not statistically significant and was attributable to two females.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs noted in pups throughout the study were comparable across groups and considered unrelated to treatment.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
- Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups:
No differences in total and live litter size or in sex ratio were noted between the control and the treated groups at birth and on Day 4 post partum.
A dose-related decrease, not statistically significant, was seen in mean litter weight at birth (25% below controls) and on Day 4 post partum (22% below controls) for the high dose females. However, this change was not considered of toxicological significance since it was due to the low number of pups in this group. No other related litter data showed differences.

- Necropsy findings in decedent pups and in pups sacri- ficed on Day 4 post partum:
No abnormalities were recorded in the decedent pups. Malrotated left hindlimb noted in one pup of the low dose group (100 mg/kg bw/day) and tip missing of the tail seen in one pup of the mid-dose group (300 mg/kg bw/day) were considered incidental and not treatment related.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Based on the results of the present study, the NOAEL for reproductive and developmental toxicity was considered to be the highest dose of 1000 mg/kg bw/day.
Executive summary:

The purpose of this study was to generate information on any toxic effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation. Experimental procedures were based on the OECD Guideline no 422 and the study was performed according to GLP.

Both male and female rats were treated for approximately 5 weeks. Three different doses were tested (100, 300 and 1000 mg/kg bw/day) and compared to a control group that received only the vehicle (corn oil).

Concerning the parents:

No differences in body weights and food consumption were observed in treated animals compared to the control group.

No clinical signs were observed during the study.No adverse findings were recorded in clinical pathology investigations (haematology, clinical chemistry and urine analysis) apart for lymphocytosis in mid- and high dose groups. No relevant differences were recorded in the absolute and relative organ weights of treated animals. No treatment-related changes were noted at macroscopic and microscopic observations.

Concerning the pups:

Fertility index and copulatory index were unaffected by treatment. Fertility index and copulatory index were unaffected by treatment and litter data parameters and sex ratio did not show differences.

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for reproductive and developmental toxicity was considered to be the highest dose of 1000 mg/kg bw/day.

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:
Sufficient for assessment
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is one study available that assesses the possible toxic effect of the test substance after repeated oral dosing. It was performed according to GLP and internationally accepted guidelines. The experimental procedures were based on the OECD guideline 422, in which both female and male rats were treated during 5 weeks. Three doses were tested (100, 300 and 1000 mg/kg bw/day) and compared to a control group that received the vehicle only.

Concerning the parents:

No differences in body weights and food consumption were observed in treated animals compared to the control group.

No clinical signs were observed during the study.No adverse findings were recorded in clinical pathology investigations (haematology, clinical chemistry and urine analysis) apart for lymphocytosis in mid- and high dose groups. No relevant differences were recorded in the absolute and relative organ weights of treated animals. No treatment-related changes were noted at macroscopic and microscopic observations.

Concerning the pups:

Fertility index and copulatory index were unaffected by treatment.Fertility index and copulatory index were unaffected by treatment and litter data parameters and sex ratio did not show differences.

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for reproductive and developmental toxicity was considered to be the highest dose of 1000 mg/kg bw/day.

There are no test reports are available that address the repeated dose toxicity of the test substance when exposure occurs via the inhalation or the dermal pathway. However, based on the outcome of the available study data, it is not deemed necessary to evaluate the other routes in more detail.


Short description of key information:
A NOAEL of 1000 mg/kg bw/d was determined for the test item based on effects observed in a combined repeated dose / reprotox screening test (OECD422).

Justification for selection of Effect on fertility via oral route:
Well documented study according to GLP and internationally accepted guidelines.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Sufficient for assessment; meets information requirements
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

Information from supporting substances has been used to fulfil the informationrequirementsfor this endpoint.For read across justification, see the atteched file in endpoint 13 ( Analogue approach justification):

Overall

Information requirements are fulfilled for this endpoint. The NOAEL for developmental toxicity was 1500 mg nonanoic acid in the rat (Celanese, 1983) which is in good agreement with an estimated NOAEL for nonanoic acid of 1820 mg/kg bw and day which was derived from a rat study using MCT (Henwood et al. 1997; MCT = Medium Chain Triglycerides; contain mainly C8 and C10 acid f from which read across to nonanoic acid can be made). MCT was also tested in the rabbit (Henwood et al. 1997). Developmental and teratogenicity was seen at the high dose only where the does showed clear maternal toxic effects (significantly reduced feed intake and reduced body weight gain p>0.01). Developmental toxicity and teratogenicity was considered to result from the low feed intake and malnutrition of the does. The NOAEL for nonanoic acid that was derived from this study was therefore only 425 mg/kg bw and day. This value should not be considered for teh derivation of a DNEL because the toxic effects were secondary to the low maternal feed intake, rather than to direct toxic effects.

The studies outlined above fulfil the information requirements for the endpoint Developmental Toxicity. There is additional information that the NOAEL for both maternal and developmental toxicity was 1500 mg/kg bw and day in a valid rat gavage study. This information is confidential and may be cited because the data requirements are already fulfilled; hence the confidential information is not needed and provides no advantage for the applicant.

For clarity, the executive summaries of all available studies are presented below.

1- In this reliable developmental toxicity study 22 female Sprague-Dawley rats were treated orally by gavage with 1500 mg/kg bw/day of the test item (purity not stated) on days 6 -15 of gestation. The 22 control animals received the vehicle corn oil. After the sacrifice at day 20 of gestation no adverse effects could be observed in the exposed dams. There were no differences between the offspring of treated and control rats with respect to teratogenic or foetotoxic effects (resorptions, viability, foetal weights, skeletal or visceral malformations or variations). Therefore the NOAEL of this study is 1500 mg/kg bw/day for dams and offspring (Celanese/Hazleton, 1983). This study was performed similar to OECD guideline 414, judged to be reliable and selected as key study (Celanese, 1983)

2 -Time-mated rats (25/group) received intravenous infusions into the caudal vein (duration 4h/day) during gestation Days 6 through 15 of a 20% lipid emulsion that contained a ratio of 3:1 of MCT and Long Chain Triglycerides (LCT) from soy bean oil at doses of 1 and 4.28 g/kg bw and day. Controls received 0.9% saline). Medium chain fatty acids are 6 to 12 carbons in length. The 20% lipid emulsion is composed primarily of 8- and 10-carbon fatty acids, with traces of 6- and 12-carbon fatty acids. The dams were observed for clinical signs and sacrificed at termination. Examinations of the dams and of the uterus content were performed.

No clinical signs or mortalities were noted. Changes in high-dose rats (enlarged lymph nodes, spleen, and renal pelvis; small thymus, small red lung foci) were considered to be related to treatment, hence the NOAEL was 1 000 mg/kg bw and day for maternal toxicity. As to the foetuses, there were no significant differences compared to controls regarding early or late resorptions, post implantation loss, dead or alive foetuses, mean uterine weight and foetal weight (though there was a trend towards lower weights at the high dose), and there were no external, soft tissue or skeletal changes that could be related to treatment. Hence, the NOAEL for developmental toxicity and teratogenicity was 4280 mg/kg bw and day (Henwood et al., 1997).

The study is considered to be valid. The intravenous route was used because the parenteral route is used in patients that cannot meet their nutritional needs by the conventional oral route, and 1000 mg/kg bw and day is the approximate clinical dose, whereas 4280 mg/kg bw and day is reportedly the highest preclinical dose that did not cause narcosis.

The study can be used for the assessment of n-nonanoic acid in a way as this has been described in the repeated dose toxicity section for the studies of Matulka and Webb who also administered triglycerides as test material. In brief,

-   the metabolism of straight chain fatty acids of 8 to 10 carbons including nonanoic acid is very similar and includes rapid degradation via ß-oxidation, which justifies cross reading form C8 and C10-fatty acids to C9 nonanoic acid.

-   The fatty acid composition of the test material of this study (Henwood et al., 1997) was almost exclusively C8 and C10, with traces of C6 and C12.

-   The mean molecular weight of the triglycerides is calculated assuming that traces are negligible, and that C8 and C10 are present in equimolar proportions (this was not specified in the publication). Then, the mean triglyceride molecular weight is 596, and the two fatty acids represent 42.5% each of the molecular weight, and also the dose, i.e. approx. 1820 mg of C8 and also of C10 fatty acid/kg bw and dose.

In a weight of evidence approach, this result can be used to assess n-nonanoic acid: NOAEL approx.. 1820 mg/kg bw and day.

3- Time-mated rabbits (15/group) received intravenous infusions into the ear vein (duration 5 h/day) during Gestation Days 7 through 19 of a 20% lipid emulsion that contained a ratio of 3:1 of MCT and Long Chain Triglycerides (LCT) from soy bean oil at doses of 1 and 4.28 g/kg bw and day. Controls received 0.9% saline. Medium chain fatty acids are 6 to 12 carbons in length. The 20% lipid emulsion is composed primarily of 8- and 10-carbon fatty acids, with only traces of 6- and 12-carbon fatty acids. The does were observed for clinical signs and sacrificed at termination, the does and of the uterus content were examined.

No treatment-related mortalities were noted. The only clinical sign was a reduced faecal output of 3/15 does on one day. Body weight change and feed intake was significantly reduced (p<0.01) in high-dose rabbits which was expected because of the high caloric test material. No adverse effect was noted in does at 1000 mg/kg bw and day, hence this was considered to represent the maternal NOAEL value.

 

Foetal findings: at the low dose (1000 mg/kg bw and day) foetal postimplantation loss was slightly increased and the mean foetal weight was slightly reduced without gaining a level of significance. This was, however, the case at the high dose where early and late resorptions were clearly increased; consequently, the number of live foetuses was decreased (p<0.01), and the mean foetal weight was significantly decreased (p<0.01). The incidence of external (p<0.05), soft tissue and skeletal malformations (p<0.05) was increased at the high dose.

The incidence of soft tissue findings was also increased in low dose foetuses, but without gaining a level of significance when individual malformations were analysed statistically. Similarly, an increase of skeletal findings was seen in the low dose group below a level of significance. Based on these findings the NOAEL for developmental toxicity was set at 1000 mg/kg bw and day, and the authors assume that the findings at 4280 mg/kg bw and day are attributable to the low feed consumption and malnutrition rather than a direct teratogenic effect of the test material (Henwood et al. 1997).

The study is considered to be valid. The intravenous route was used because the parenteral route is used in patients that cannot meet their nutritional needs by the conventional oral route, and 1000 mg/kg bw and day is the approximate clinical dose, whereas 4280 mg/kg bw and day is reportedly the highest preclinical dose that did not cause narcosis.

The study can be used for the assessment of n-nonanoic acid in a way as this has been described in the repeated dose toxicity section for the studies of Matulka and Webb who also administered triglycerides as test material. In brief,

-   the metabolism of straight chain fatty acids of 8 to 10 carbons including nonanoic acid is very similar and includes rapid degradation via ß-oxidation, which justifies cross reading form C8 and C10 fatty acids to C9 nonanoic acid.

-    The fatty acid composition of the test material of this study (Henwood et al., 1997) was almost exclusively C8 and C10, with traces of C6 and C12.

-     The mean molecular weight of the triglycerides is calculated assuming that traces are negligible, and that C8 and C10 are present in equimolar proportions (this was not specified in the publication). Then, the mean triglyceride molecular weight is 596, and the two fatty acids represent 42.5% each of the molecular weight, and also of the dose, i.e. approx. 425 mg of C8 and also of C10 fatty acid/kg bw and dose.

In a weight of evidence approach, this result can be used to assess n-nonanoic acid: NOAEL approx. 425 mg/kg bw and day.

 

 4- According to the CLH report, the NOAEL of nonanoic acid was 1500 mg/kg bw and day for both maternal and developmental toxicity in a valid developmental study using rats. It may also be acknowledged that a developmental toxicity study with Nonanoic acid was submitted in the context of the BPD 98/8/EC Annex I inclusion procedure. The study is owned by the respective applicant W. Neudorff GmbH KG and the data are protected. However since the data requirement for the evaluation of developmental toxicity is fulfilled with the references provided above and the study is not used to the advantage of the applicant of Decanoic and Octanoic acid (Fatty Acid Consortium) it may be cited and discussed also for the evaluation of Decanoic acid and Octanoic acid (Austria, 2012: CLH reports for Octanoic and Decanoic acid, respectively, section 4.11.3).

The Competent Authority further argued that animals and humans are exposed to fatty acids via food feed, that fatty acids are rapidly and completely metabolised to carbon dioxide and water, that no bioaccumulation has to be considered, and that no adverse effects were seen in repeated dose studies even at high doses of MCT, and that no adverse effects of fatty acid ingestion on reproduction is known. Based on this it was concluded that no further developmental toxicity study would be required (Austria 2011; section 4.11). EFSA (2013) argued likewise that no new studies are necessary on this endpoint. See also assessment reports included in Section 13.

 

 


Justification for selection of Effect on developmental toxicity: via oral route:
Oral rat study, comparable to guideline study with acceptable restrictions

Justification for classification or non-classification

No further studies required. Based on the available information a classification of the test substance is not required according to Regulation (EC) No 1272/2008 as well as to the Council Directive 67/548/EEC.