2-ethyl-2-[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been conducted in according to OECD guideline and GLP without deviation

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:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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.

Key result

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:

other: both for general toxicity, reproductive and developmental toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

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.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

sexual maturation

clinical signs

mortality

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

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

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

Description of key information

There is one study available describing developmental toxicity effects (Azuka and Daston, 2014) on the read-across substance Trimethylolpropane Caprylate Caprate (CAS11138-60-6). Based on the results of that study there is no indication of any potency to cause developmental effects as the determined NOAEL for developmental effects was > 2000 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions; few details on test substance given, no analysis of the test compound

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

few details on test substance given, no analysis of the test compound

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: young adult
- Weight at study initiation: Mean of the maternal body weight: 226 g (Vehicle), 225 g (200 mg/kg bw/day), 227 g (600 mg/kg bw/day), 226 g (2000 mg/kg bw/day)
- Fasting period before study: No
- Housing: Virgin females were cohabitated with singly-housed male rats, one male per female rat for a maximum of 5 days and returned to individual housing in stainless steel wire-bottomed cages after mating.
- Diet: Certified Rodent Diet No. 5002 (PMI Feeds Inc. St.Louis, MO), ad libitum
- Water: water passaged through a reverse osmosis membrane with chlorine added as a bacteriostat, ad libitum
- Acclimation period: yes, period not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

dermal

Vehicle:

corn oil

Details on exposure:

- Doses: 0, 200, 600, and 2,000 mg/kg/day.

- Dose formulation: 0 (vehicle only), 100, 300, and 1,000mg/mL

- Dosage volume: 2 mL/kg.

- Application of dose: The dosage amount was applied directly to the clipped area on the dorsum of the rat at approximately the same time each day and spread uniformly over the area with a glass rod. The skin application site was occluded during treatment with a gauze pad secured with Vetrapt or Micropores tape to prevent oral ingestion and to minimize loss of material from under the patch.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Treatment on Gestation Days (GD) 6 - 15

Frequency of treatment:

daily

Duration of test:

Termination of the study by CO2 inhalation on GD 20.

No. of animals per sex per dose:

25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose dependent occurrence of skin irritation. Higher levels than 2000 mg/kg bw/day might be expected to produce marked irritation thereby compromising the interpretaion of developmental results.
- Rationale for animal assignment (if not random): Computer-generated randomization by weight (Barlett´s test for homogeneity) such that the groups were not statistically different (5% significance level) from each other.

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: Animals were checked for mortality twice daily during the treatment period and daily thereafter.


DETAILED CLINICAL OBSERVATIONS
- Time schedule: Animals were checked for signs of reaction to treatment and/or symptoms of illness once daily before treatment, approx. 60 min after treatment during the dosing period. The dosing site was examined daily prior to substance application for signs of skin irritation according to Draize.


BODY WEIGHT
- Time schedule for examinations: Recorded on GD 0 and daily during the treatment period.


FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg b.w./day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day # 20
- Organs examined: The uterus, uterine contents, position of the fetuses in the uterus and number of corpora lutea. Number and distribution of intrauterine implantations were classified as live or death fetuses, late intrauterine deaths (resorptions) and early intrauterine resorption sites. Live fetuses were sexed and further examined (see below).

Ovaries and uterine content:

- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions

Fetal examinations:

- External examinations: all per litter
- Soft tissue examinations: half per litter
- Skeletal examinations: half per litter
- Head examinations: half per litter (the heads of the animals used for soft tissue examinations)

Statistics:

Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution (Snedecor and Cochran, 1967). Quantitative continuous data (e.g., maternal body weights, body weight changes, feed consumption values, litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights, fetal anomaly data, and fetal ossification data) were analyzed using Bartlett’s Test
for Homogeneity of Variance (Sokal and Rohlf, 1969) and the Analysis of Variance (Snedecor and Cochran, 1967) when Bartlett’s Test was not significant (p40.05). If the Analysis of Variance was significant (pr0.05), Dunnett’s Test (Dunnett, 1955) was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate, i.e., Bartlett’s Test was significant (pr0.05), the Kruskal-Wallis Test (Sokal and Rohlf, 1969) was used when < 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p<0.05), Dunn’s Method of Multiple Comparisons (Dunn, 1964) was used to identify the statistical significance of the individual groups. If there were 475% tied, Fisher’s Exact Test (Siegel, 1956) was used to analyze the data. Count data obtained at Caesarian-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: local irritation

Details on maternal toxic effects:
The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or feed consumption. Two animals in the control group and one animal in the high-dose group died within 6 h after first application; these were not considered to be treatment related and the animals were replaced. One dam of the mid-dose goup (1/25) having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility.
Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar (local alopecia, chromorhinorrhea, and neck lesions).

Key result

Dose descriptor:

NOAEL

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:

There were no significant differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. The observed effects in fetuses were dose-independent and regarded to be sporadic.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

changes in postnatal survival

external malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Skin reaction observations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Maximum possible incidences^a	375/25	375/25	375/25	375/25
Erythema
Total	0/0	2/1	22/4	91/13^b
Grade 1	0/0	2/1	10/4	81/13^b
Grade 2	0/0	0/0	4/1	10/4^b
Flaking
Total	11/3	15/2	55/6	170/17^b
Grade 1	11/3	9/2	27/5	61/14^b
Grade 2	0/0	6/1	19/4	71/14^b
Grade 3	0/0	0/0	9/1	38/7 ^b
Edema
Total	0/0	0/0	23/4	83/11^b
Grade 1	0/0	0/0	18/4	59/11^b
Grade 2	0/0	0/0	5/1	24/6^b
Scab	0/0	0/0	6/2	19/4

a:       Maximum incidence : Days x rats from first treatment on GD 6 through sacrifice on GD 20 divided by the number of rats examined per group on GD 6-20

b:        Significantly different from vehicle control group value (p≤0.01)

 

Table 2: Maternal reproductive, litter, and fetal alteration observations: Caesarian-Section results on GD 20

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Rats pregnant and sectioned on Day 20 of gestation (n)	25	23	22^b	24
Corpora lutea/dam	16.4	16.6	16.9	16.5
Implantation sites/litter	15.0	15.4	14.9	14.2
Litter size
Live fetuses/litter	14.6	14.6	14.0	13.3
Live fetuses (n)	364	335	308	320
Dead fetuses (n)	0	0	0	0
Resorptions	0.4	0.9	0.9	0.9
Early (n)	10	20	19	21
Late (n)	1	0	0	0
Dams with any resorptions n(%)	9 (36)	11 (48)	15 (68)	11 (46)
% resorbed/litter	2.9	5.4	5.8	5.0
% male/litter	51.3	50.8	48.1	47.7
Live fetal body weight (g/litter)	3.68	3.62	3.69	3.75
Male	3.77	3.68	3.82	3.85
Female	3.58	3.56	3.58	3.65
Fetuses evaluated (n)	364	335	308	320
Litters with any alterations observed n(%)	10 (40)	8 (35)	14 (64)	7 (25)
Fetuses with any alterations observed n(%)	13 (3.5)	10 (3.0)	20 (6.5)	9 (2.0)
% fetuses/litter with any alterations observed	3.5	2.9	6.8^c	2.7

b:       Excludes values for one dam, which had a litter consisting of seven early resorptions.

c:       Significantly different from vehicle control group value (p≤0.05)

Table 3: Fetal evaluations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Litters evaluated	25	23	22^b	24
Fetuses evaluated	364	335	308	320
Live	364	335	308	320
Fetal gross external alterations	364	335	308	320
Tail: kinked
Litter incidence, n (%)	0(0)	1 (4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.3)	0(0)	0(0)
Body: hematoma
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	0(0)
Fetal incidence, n (%)	1 (0.3)	0(0)	0(0)	0(0)
Fetal soft tissue alterations, evaluations	174	162	149	155
Vessels: umbilical artery descended to the left of urinary bladder
Litter incidence, n (%)	2(8.0)	3(13.0)	2(9.1)	2(8.3)
Fetal incidence, n (%)	2(1.1)	3(1.8)	3(2.0)	2(1.3)
Vessels: apparent additional umbilical artery descended left of the bladder
Litter incidence, n (%)	0(0)	0(0)	1(4.5)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	1(0.7)	0(0)
Fetal skeletal alterations, evaluations	190	173	159	165
Cervical vertebrae: cervical rib present at 7th cervical vertebrae
Litter incidence, n (%)	2(8.0)	1(4.3)	1(4.8)	0(0)
Fetal incidence, n (%)	2(1.0)	2(1.2)	1(1.2)	0(0)
Thoracic vertebrae: centrum, bifid
Litter incidence, n (%)	1(4.0)	1(4.3)	5(22.7)	0(0)
Fetal incidence, n (%)	1(0.5)	1(0.6)	5(3.1)^a	0(0)
Lumbar vertebrae: centrum, bifid
Litter incidence, n (%)	0(0)	1(4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.6)	0(0)	0(0)
Ribs: wavy
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	1(4.2)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	1(0.5
Sternal centra: 1st, not ossified
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	2(8.3)
Fetal incidence, n (%)	1(0.5)	0(0)	0(0)	2(1.3)
Sternal centra: 1st, incompletely ossified
Litter incidence, n (%)	3(12.0)	3(13.0)	2(5.1)	1(4.2)
Fetal incidence, n (%)	4(2.1)	4(2.3)	2(1.2)	1(0.6)
Pelvis: pubis, incompletely ossified
Litter incidence, n (%)	3(12.0)	0(0)	4(18.2)	3(12.5)
Fetal incidence, n (%)	3(1.6)	0(0)	5(3.1)	3(1.8)
Pelvis: ischium, incompletely ossified
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	0(0)

a: Significantly different from vehicle control group (p≤0.01)

Conclusions:

TMPCC did not cause any developmental toxicity in the Sprague-Dawley rat at dermal dosages up to 2,000 mg/kg/day.

Executive summary:

The developmental toxicity potential of trimethylolpropane caprylate caproate (TMPCC, CAS no. 11138-60-6) was evaluated in rats. Sprague-Dawley rats were administered TMPCC in a corn oil suspension dermally at dose levels of 0, 200, 600, or 2,000 mg/kg/day on gestation days (GD) 6–15 (sperm positive day5GD 0). Caesarean sections were performed on GD 20 and fetuses were evaluated for viability, growth, and external, visceral, and skeletal abnormalities. Each group consisted of 25 females, with at least 22 per group being pregnant. The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain. There were no differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. TMPCC did not cause any developmental toxicity in the Sprague-Dawley rat at dermal dosages up to 2,000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Read-across

 

From Decanoic acid, ester with 2-ethyl-2(hydroxymethyl)-1,3-propanediol octanoate CAS11138-60-6

 

·Carbon number in Fatty Acids: C8, 9     

·Carbon number in Polyol: C6

·Total Carbons in Polyol Ester: C24     

·Molecular Weight: 415     

 

To Nonanoic acid, triester 2-ethyl-2(hydroxymethyl)-1,3-propanediol with CAS126-57-8

  

·Carbon number in Fatty Acids: C9     

·Carbon number in Polyol: C6

·Total Carbons in Polyol Ester: C33     

·Molecular Weight: 555   

Both belong to the Trimethylolpropane (TMP) Esters as they have the trimethylolpropane group as a common structural part

 

For both substances, it is valid to assume that when metabolism of these polyesters takes place, this firstly leads to the generation of the corresponding fatty acids and the polyol alcohol.

For substance “Decanoic acid, ester with 2-ethyl-2(hydroxymethyl)-1,3-propanediol octanoate” (CAS11138-60-6) this relates to trimethylolpropane and 2 C10 fatty acid chains. For substance “Nonanoic acid, triester 2-ethyl-2(hydroxymethyl)-1,3-propanediol” (CAS126-57-8) this also relates to trimethylolpropane and 3 C9 fatty acid chains.

As it is not expected that the developmental toxicity is different between C10 and C9 fatty acid chains read-across is considered justified.

Justification for selection of Effect on developmental toxicity: via dermal route:
There is only one study available (Azuka and Daston, 2014) on the read-across substance Trimethylolpropane Caprylate Caprate.

Toxicity to reproduction: other studies

Description of key information

Qsar evaluations according to IRFMN ( Estrogen Receptor relative binding affinity model (IRFMN) (Version 1.0.0)) and IRFMN/CERAPP ( Estrogen Receptor-mediated effect (IRFMN/CERAPP) (version 1.0.0)) reveal that at an endocrine disruptor screening the target substance results inactive.

Link to relevant study records

Reference

Reference 1

Endpoint:

toxicity to reproduction: other studies

Remarks:

Estrogen Receptor Relative Binding Affinity model (IRFMN) (version 1.0.1)

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Dose descriptor:

other: Activity

Basis for effect level:

other: Qsar evaluation

Remarks on result:

not determinable due to absence of adverse toxic effects

This constituent resulted inactive

Compound SMILES: CCCCCCCCC(=O)OCC(CC)(COC(=O)CCCCCCCC)COC(=O)CCCCCCCC

Experimental value: -

Predicted activity: Inactive

Classification tree final node: 12

Reliability: the predicted compound is into the Applicability Domain of the model

Remarks: None

Conclusions:

The constituent resulted inactive

Reference 2

Endpoint:

toxicity to reproduction: other studies

Remarks:

Estrogen Receptor-mediated effect (IRFMN/CERAPP) (version 1.0.0)

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Qualifier:

according to guideline

Guideline:

other: IRFMN-CERAPP model

Version / remarks:

1.0.0

Deviations:

not applicable

Principles of method if other than guideline:

Estrogen Receptor mediated effect classification model for endocrine disruptor screening, based on the the dataset from the CERAPP project (U.S. EPA).

Type of method:

other: Qsar

Dose descriptor:

other: Activity

Basis for effect level:

other: Qsar evaluation

Remarks on result:

not determinable due to absence of adverse toxic effects

Conclusions:

The substance resulted inactive

Justification for classification or non-classification

Based on the results of the combined reprotox / repeated dose toxicity testing and developmental toxicity tests, the test item should not be classified for reproductive effects according to the criteria described in Directive 67/548/EEC (Dangerous Substances Directive) and the EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) respectively.