Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral: OECD 422, rat, NOAEL fertility1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
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
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2 due to read-across) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for analogue read-across

There are no data on the reproduction toxicity of Pentaerythritol, mixed esters with linear and branched fatty acids. The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Toxicity to Reproduction

126-57-8

A combination Repeated Dose/Reproduction/Developmental Toxicity Screening test was performed with Prenatal Developmental Toxicity Study was performed with 2-ethyl-2-[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate (CAS RN 126-57-8) via the oral route equivalent or similar to OECD 422 (Salvador, 2015). 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 using male and female Sprague-Dawley rats. 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). 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 spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. 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. The copulatory and fertility indices were comparable between control and treated groups. 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. Fertility index and copulatory index were unaffected by treatment and litter data parameters and sex ratio did not show differences. Based on the results, 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.

 

90-day oral, dermal and inhalation repeated dose toxicity studies, performed with three source substances, are available (Cruzan, 1988; Dulbey, 1992; Müller, 1998). In these studies no treatment-related effects were observed on the weight and histopathological structure of reproductive organ and tissues.

 

A waiver for the extended one-generation reproductive toxicity study (EOGRTS) was included. The results of the studies performed on source substances, which cover a wide range of reproductive, fertility and developmental parameters, did not show any potential for toxicity to reproduction (fertility) in rats and a potential for developmental toxicity only at high dose level. It is reasonable to assume for the target substance, that the (lack of) effects noted in the available studies will reflect the results of reproductive parameters assessed specifically in an extended one-generation reproduction toxicity study. Therefore performing an extended one-generation reproduction toxicity study (standard configuration or with additional modules)is not scientifically necessary and, considering concerns regarding the use of vertebrate animals for experimental purposes, unjustified.

 

Short description of key information:

Oral: OECD 422, rat, NOAEL fertility1000 mg/kg bw/day

 

Justification for selection of Effect on fertility via oral route:

Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information

NOAEL for developmental toxicity is considered to be1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(only 15 presumed pregnant females per group, exposure on day 0-19 of gestation, only 2 dose levels, nonstandard dermal exposure, limited details on exposure)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, N.Y.
- Age at study initiation: approx. 9 weeks
- Mean weight at study initiation: 248 g
- Diet: Purina Certified Rodent Chow #5002 (Meal), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 40 - 60
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Type of wrap if used: open exposure, no wrap
- Time intervals for shavings or clipplings: no data on frequency; clipped, intact skin
- Site: dorsal
Controls: The rats of the control group were clipped and collared. The intact dorsal skin of each rat was stroked with the tip of a syringe, but no test material was applied.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The amount of the test material applied with a syringe was calculated based on the body weight of the animals and the density of the test substance.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes. To minimize ingestion of the test material, the rats were fitted with cardboard Elizabethan-style collars.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
gestation days 0 - 19
Frequency of treatment:
daily
Duration of test:
The animals were sacrificed on day 20 of gestation.
Remarks:
Doses / Concentrations:
800 and 2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
15 presumed-pregnant females
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on results of a 13-week dermal study previously conducted with the same material
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: signs of pathosis, abortion, premature delivery, and death

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 3, 6, 10, 13, 16, and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for calculation: days 0-3, 3-6, 6-10, 10-13, 13-16, and 16-20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracic and abdominal cavities were exposed and all organs were examined grossly for evidence of pathosis.

OTHER:
- Clinical chemistry: alanine aminotransferase (ALT), albumin, albumin/globulin ration, alkaline phosphatase (ALP), aspartate aminotransferase (AST), bilirubin, calcium, chloride, cholesterol, creatinine, globuline, glucose, iron, lactate dehydrogenase (LDH), phosphorus, potassium, sodium, sorbitol dehydrogenase (SDH), total protein, triglycerides, urea nitrogen, and uric acid
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
- Number of live and dead fetuses: Yes
- Other: The ovaries of non-pregnant females were grossly examined and then discarded.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
- Analysis of variances and group comparison using Fisher's Exact or Dunnett's test (maternal biophase and cesarean section data, and fetal data)
- ANOVA and Fisher's Exact test (fetal skeletal data)
- Fisher's Exact test (fetal visceral data)
- SAS procdures, Student-Newman-Keul's multiple comparison test (clinical chemistry data)
P < 0.05
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: slight local effects

Details on maternal toxic effects:
- General observations: neck lesions, red nasal exudate, and chromodacryorrhea in all groups (considered not to be test substance-related as these signs are common in animals that are collared)
- Local effects: mild dermal irritation including erythema and flaking of the skin in the treatment groups
- Body weight: similar to controls in both treatment groups
- Body weight gain: similar to controls in both treatment groups
- Uterine and net body weights: similar to controls in both treatment groups
- Food consumption: similar to control in both treatment groups; only difference (statistically significant) in high dose group on day 13-16: 31.5 g vs. 29.5 g (corresponding control data)
- Necropsy: no remarkable findings were observed
- Fetal status and uterine position: no parameter evaluated appeared to be adversly affected
- Clinical chemistry: no differences between treated and non-treated rats were observed
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Fetal body measurements: Mean fetal body weights and crown-rump lengths, parameters of body growth and development were normal compared to controls.
- Fetal examination: No malformations or variations were observeed. Bruises were observed on the skin of 4 fetuses from the control group and 2 fetuses form the 800 mg/kg bw/day group (considered to be incidental). One fetus from one dam exposed to 800 mg/kg bw/day was pale in colour. No other remarkable findings were observed during external examination.
- Malformations (lumbar and sacral vertebrae, tail, sternebrae, and ribs) were observed. The incidence was low and there was no apparent dose-response relationship, these effects were considered to be not treatment-related. Comparable incidences of variant skeletal development were observed in both cotnrol and trated fetuses.
- Visceral examinations: A statistically significant (high dose) increase in the number of fetuses with levocardia was observed. The response appeared to be dose-related
Levocardia:
Litters: control: 0 (0%); 800 mg/kg bw/day: 2 (14.3%); 2000 mg/kg bw/day: 7 (50%); 14 litters per group examined
Fetuses: control: 0 (0%); 800 mg/kg bw/day: 3 (3.2%); 2000 mg/kg bw/day: 7 (10.1%); 94, 93, and 99 fetuses examined, respectively
Microphthalmia, anophtalmia and "apparent" hydronephrosis were also observed. Variant visceral development was observed in control and treated fetuses.
Dose descriptor:
NOAEL
Effect level:
< 800 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
visceral malformations
Abnormalities:
not specified
Developmental effects observed:
not specified

Levocardia was the only parameter affected in fetuses of dams treated with the test substance during gestation. In other studies, levocardia was observed in control fetuses, too. However, the effect of the test substance on heart development should be focused on in further studies as well as the impact, this effect has on postnatal survival.

Conclusions:
In a developmental toxicity study, pregnant rats were dermally exposed to the test substance. No adverse effects were observed in any maternal or reproductive parameter nor on external and skeletal development of fetuses. Levocardia was observed in 3.2% and 10.1% of the fetuses exposed in utero to 800 and 2000 mg/kg bw /day, respectively. Thus, the developmental NOAEL was determined to be < 800 mg/kg bw.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2 due to read-across) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
800 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Additional information

Justification for analogue read-across

Data on developmental toxicity of Pentaerythritol, mixed esters with linear and branched fatty acids are not available. The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Developmental toxicity/teratogenicity

CAS 67762-53-2

The potential of Fatty acids, C5-9, tetraesters with pentaerythritol to cause developmental toxicity was assessed in a prenatal developmental toxicity study performed according to a protocol adapted from OECD guideline 414 (Feusten, 1988). 15 pregnant dams/dose were exposed to 800 and 2000 mg/kg bw/day via the dermal route during gestation day 0 to 19. The test substance was applied to the clipped skin and left uncovered. To minimize ingestion of the test substance, the rats were fitted with cardboard Elizabethan-style collars. On day 20 of gestation the animals were euthanized and examined. Neck lesions, red nasal exudate, and chromodacryorrhea was noted in all groups, but considered not to be test substance-related as these clinical signs are common in animals that are collared. No toxicologically relevant effects were observed on body weight (gain), food consumption and clinical chemistry parameters in the P-females. Mild dermal irritation, including erythema and flaking of the skin, was observed at the application site of the treated animals. No treatment-related effects on the dam reproductive parameters (gravid uterus weight, number of corpora lutea, number of implantation sites, pre-implantation loss, early resorptions) were observed. The NOAEL for maternal systemic toxicity was set at1000 mg/kg bw/day. There were no significant differences between the control and treatment pups in the developmental parameters (post-implantation loss, body weight, sex ratio, viability). The incidence of malformations and variations in the offspring was comparable between the control and treatment groups. A dose-dependent number of offspring had levocardia, although no heart malformations were observed. In the litters the incidence was 0 (0%), 2 (14.3%) and 7 (50%) in the control, low-dose and high-dose group, respectively (14 litters per group examined). In the foetuses the incidence was 0 (0%), 3 (3.2%) and 7 (10.1%) in the control, low-dose and high-dose group, respectively (94, 93, and 99 fetuses examined, respectively). Levocardia has been observed in vehicle control foetuses (Smith et al. 1988) and in the control foetuses conducted in the test laboratory, meaning there is a natural background incidence. The NOAEL for embryo-/fetotoxicity and teratogenicity in rats was considered to be < 800 mg/kg bw/day and the LOAEL = 800 mg/kg bw/day.

 

CAS 11138-60-6

The potential of Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol to cause developmental toxicity was assessed in a prenatal developmental toxicity study performed according to OECD guideline 414 and under GLP conditions (Azuka and Daston, 2004). 25 pregnant dams/dose were exposed to 200, 600 and 2000 mg/kg bw/day of the test substance via the dermal route during gestation day 6 to 15. The test substance was applied to the clipped skin for 6 hours/day under occlusive conditions. On day 20 of gestation the animals were euthanized and examined. Two animals in the control group and one animal in the high-dose group died within 6 hours after first application. These deaths were not considered to be treatment-related and the animals were replaced. No treatment-related effects were observed on body weight (gain) and food consumption in the P-females. The mid- and high-dose levels caused some local skin irritation at the site of application. One dam of the mid-dose group (1/25) had seven early resorptions. This is considered to be an incidental occurrence as there was no dose-dependency and the number was within the historical control range of the test facility. No treatment-related effects on the dam reproductive parameters (number of corpora lutea, implantation sites, pre-implantation loss, resorptions) or on reproductive tissues and organs were observed. The NOAEL for local effects in considered to be 200 mg/kg bw/day. The maternal NOAEL for systemic effects is set at2000 mg/kg bw/day.

 

There were no significant differences between the control and treatment pups in the developmental parameters (post-implantation loss, body weight, sex ratio, viability). The percentage of fetuses/litter with alteration(s) and the percentage of fetuses with bipartite ossification of the thoracic vertebra were significantly increased in the mid-dose group. Because the incidence was not increased in the high-dose group, and as there was no significant difference between treatment and control groups in the percentage of litter with alterations and percentage of foetuses with alteration, this is considered to be an incidental occurrence. The NOAEL for developmental toxicity and teratogenicity was considered to be2000 mg/kg bw/day.

 

A waiver for the prenatal developmental toxicity study in a second species was included. Information is available from developmental toxicity studies in rats performed with source substances. Considering the results of all the studies and taking into account the fact that for the only observed effect - the increase in levocardia - treatment relation and the toxicological relevance can be questioned it is concluded that the registered substance is considered to have a low potential for developmental toxicity and teratogenicity. Furthermore, the preliminary results of a comparative analysis of data on pharmaceutical compounds suggest that the 2nd species does not add significant information for the assessment of developmental effects. For substances for which there is no convincing data indicating a potential difference in relative species sensitivity between the rat and rabbit, performing a developmental toxicity study in a 2nd species would not be justifiable for animal welfare reasons. Therefore, performing a prenatal developmental toxicity study in a 2nd species is considered not to add new information for hazard assessment and therefore is scientifically and, considering concerns regarding the use of vertebrate animals for experimental purposes, morally unjustified.

 

Overall conclusion for developmental toxicity/teratogenicity

There are no available studies on the developmental toxicity and teratogenicity of Pentaerythritol, mixed esters with linear and branched fatty acids. Therefore analogue read-across from source substances administered via the oral and dermal route was applied. Due to the bulkiness and high molecular weight of the target substance, the absorption percentage via oral route is expected to be higher than via the dermal route, indicating the results via the oral route give more accurate indications of the systemic effect. Therefore no hazard was identified for developmental toxicity.

The overall NOAEL for developmental toxicity is considered to be1000 mg/kg bw/day.

 

Justification for selection of Effect on developmental toxicity: via oral route:

Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

 

Justification for selection of Effect on developmental toxicity: via dermal route:

Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study performed via the dermal route based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Pentaerythritol, mixed esters with linear and branched fatty acids, data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Additional information


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