1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-01-2022 to 02-03-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Version: 2018

Deviations:

yes

Remarks:

Only 15 litters in the Mid dose group at scheduled necropsy; however, this was considered to be related to the increased rate of abortions and there were sufficient litters for examination in the remaining groups

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Version: 1998

Deviations:

yes

Remarks:

Only 15 litters in the Mid dose group at scheduled necropsy; however, this was considered to be related to the increased rate of abortions and there were sufficient litters for examination in the remaining groups

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters

EC Number:

290-754-9

EC Name:

1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters

Cas Number:

90218-76-1

Molecular formula:

C33H51O6 to C39H66O6

IUPAC Name:

tris(mixed decyl and octyl)benzene-1,2,4-tricarboxylate

Test material form:

liquid

Remarks:

Nearly colourless (at most slightly yellowish/yellow-stitched)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL:
Source and lot/batch number of test material: Sponsor (Sasol Germany GmbH Anckelmannsplatz 1, 20537 Hamburg, Germany); Lot/Batch# 05804/MA
Expiration date of the lot/batch number: 31 January 2023

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
Storage condition of test material:Controlled room temperature (15-25°C, ≤70% relative humidity, protected from UV-light and direct sunlight).
Stability under test conditions: Stability of the test item in the vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to Charles River Laboratories Hungary Kft.) using an analytical method (High-Performance Liquid Chromatography method with UV Detector (HPLC-UV), study code 21/019-316ANE)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS:
Appearance:Nearly colourless (at most slightly yellowish/yellow-stitched), liquid
Purity: 98.8 %
EC No.: 290-754-9
CAS No.: 90218-76-1

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S&K-Lap Kft. (Address: 2173 Kartal, Császár út 135, Hungary).
- Age at study initiation: Young adult female rabbits, nulliparous and non-pregnant, approximately 4 months old at insemination.
- Weight at study initiation: 3482 - 4071g at onset of the treatment, did not exceed ± 20% of the mean weight at onset of treatment.
- Fasting period before study: Not specified
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbits in adjoining cages.
- Diet (e.g. ad libitum): The animals received ad libitum quantities of a standard diet for rabbits produced by Cargill Takarmány Zrt. (Address: H-5300 Karcag, Madarasi road, Hungary), (Batch number: 0008019204/ 0008038307, Expiry date: 06 April 2022 / 13 April 2023).
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum.
- Acclimation period: 5-13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.7 – 26.3°C (target range: 15 - 21°C)
- Humidity (%): 27 - 67% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 2022-01-16 To: 2022-02-15

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the selected vehicle (corn oil) as a visibly stable suspension at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of the Test Facility. The formulations were stirred with a magnetic stirrer from the time of preparation until completion of each treatment. A constant volume of 1.5 mL/kg body weight was administered to all test animals. The individual volume of the treatment was based on the most recent individual body weight of each animal. Considering the declared purity of 98.8%, no correction for purity of the test item was applied during formulation. Formulations were prepared fresh every day prior to administration to animals.

Stability of the test item in the vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to Charles River Laboratories Hungary Kft.) using an analytical method (High-Performance Liquid Chromatography method with UV Detector (HPLC-UV), study code 21/019-316ANE).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of the trial formulation, corn oil was selected as the vehicle for this study in agreement with the Sponsor.
- Concentration in vehicle: 0, 66.7, 200 or 666.7 mg/mL for the 0, 100, 300, and 1000 mg/Kg bw/day dose levels, respectively.
- Amount of vehicle (if gavage): 1.5 mL/Kg body weight
- Lot/batch no. (if required): Source: Sigma-Aldrich Co. Batch number:MKCM9808 / MKCN9742
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of control (vehicle) and test item formulations for concentration and homogeneity were performed within the determined stability period at the Test Facility under the control of the Analyst. Representative samples of control (vehicle) and test item formulations were analysed two times during the study, in the first and last weeks of dosing.

On each sampling occasion, two sets of top, middle and bottom duplicate samples were taken from test item formulations for homogeneity and concentration measurement, one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Analyses of the formulations for concentration and homogeneity of test item were performed on each occasion using a validated analytical method (Gas Chromatography system with Flame Ionization Detector (GC-FID)) in the Analytical Department of the Test Facility (CRL Study code: 21/019-316AN [1]).

Acceptance criterion of the concentration analysis was 100 ± 15% of the nominal concentration.
Acceptance criterion of the homogeneity was that the RSD (relative standard deviation) of replicates (top, middle and bottom of test item formulations) were less than 10%.

The formulation analysis was conducted under the control of the responsible Analyst in compliance with the relevant Standard Operation Procedures of the Test Facility.

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Insemination procedure: Synchronisation of the oestrus cycle of the does was initiated 48 hours prior to insemination by administration of PMSG (gonadotropin) hormone (40 IU/female, sc). Insemination was performed in batches by the breeder at the Test Facility, with sperm originated from New Zealand White male rabbits from the same source as the females. Each female was inseminated with diluted sperm containing at least 2 million spermatozoa. At the same time as the artificial insemination was performed, ovulation was stimulated with 1 mL buserelin-based compound (0.2 mL/animal, i.m.). The day of insemination was regarded as Gestation Day 0 (GD0).
- Any other deviations from standard protocol: Not specified
The inseminated, assumed pregnant female rabbits were randomly allocated to the test groups on each insemination day in such a way that the group averages of the body weight were as similar as possible. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups to ensure that animals of all test groups were as nearly as practicable of a uniform weight.

Duration of treatment / exposure:

Daily from gestation day 6 (GD 6) to gestation day 27 (GD 27)

Frequency of treatment:

Daily

Duration of test:

GD 0 to GD 28

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 - Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2 - Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3 - Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4 - High dose

No. of animals per sex per dose:

22 inseminated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route was selected as it is the most relevant route of potential human exposure.
The dose levels were set by the Sponsor / Study Monitor based on the available data (results of a Dose Range Finding (DRF) study (CRL Hungary Study code: 21/019-105NE)). Considering that in the DRF study there were no test item related adverse effects at 1000 mg/kg bw/day, this dose level was selected as the High dose for this study. Lower doses are spaced with a factor of ~3.

- Rationale for animal assignment: The inseminated, assumed pregnant female rabbits were randomly allocated to the test groups on each insemination day in such a way that the group averages of the body weight were as similar as possible. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups to ensure that animals of all test groups are as nearly as practicable of a uniform weight.

- Fasting period before blood sampling for (rabbit) dam thyroid hormones: N/A

- Time of day for (rabbit) dam blood sampling: N/A

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day, on GD0 only one inspection was conducted). General clinical observations were made twice daily (at the beginning and end of each working day). Only one general clinical observation was made on the first day (in the afternoon), on the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, clinical observation (detailed) was made only once on necropsy days (in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals at the start (GD 0), at the onset of treatment (GD 6), then weekly and on the day of necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical.
Pertinent behavioural changes and all signs of toxicity, including mortality, were recorded including onset, degree and duration of signs as applicable. Signs evaluated included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Body weight gain was calculated for each interval, including GD 0-6, GD 6-27 and GD 0-27.

FOOD CONSUMPTION (if feeding study): Yes
- Food consumption was measured on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Food consumption was calculated for each interval, including GD0-3, GD3-6, GD 0-6, GD6-9, GD9-12, GD12-15, GD15-18, GD18-21, GD21-24, GD24-27, GD 6-27 and GD 0-27.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD 28
- Organs examined: All animals (including those that died during the test or were removed from the study for animal welfare reasons) were subjected to a necropsy and a macroscopic examination.

Animals were killed by pentobarbital anaesthesia (intramuscular injection of Euthanimal 40% (400 mg/mL sodium pentobarbital solution)) followed by exsanguination.

Caesarean section was performed on GD 28 in surviving does. The ovaries and uterus were removed, and the pregnancy status ascertained. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appear non-gravid were further examined to confirm the non-pregnant status (i.e. by Salewski staining or a suitable alternative method). The corrected body weight of each doe was calculated (body weight on GD28 minus weight of the gravid uterus).

The ovaries and uterus were examined - the number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic deaths and foetal deaths were counted, the number and percent of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

The weight of the thyroid gland with parathyroid glands was measured for all surviving does and the thyroid plus parathyroids were retained, embedded in paraffin wax and sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Organ weights were not measured for animals found dead during the study.

Ovaries and uterine content:

The ovaries and uterine content were 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

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: N/A
After ensuring humane death, each fully developed foetus was weighed individually. The crown-rump length of foetuses was measured. All foetuses were externally and viscerally examined, and sex was determined (internal sex organs).
Special attention was paid to incomplete testicular descent or cryptorchidism for male foetuses.

The heads from approximately half of the animals from each litter were removed and processed for evaluation of soft tissue alterations (including eyes, brain, nasal passages and tongue), using fixation in Sannomiya mixture for Wilson-sections. After fixation the head was examined by Wilson's free-hand razor blade method. All foetuses were prepared for skeletal examination. The skeletons were examined after double staining with acetic Alcian blue + alkalic Alizarin red (cartilage and bone staining). All abnormalities (external, visceral and skeletal malformations and variations) found during the foetal examinations were recorded; photographic records were made additionally where the Study Director considered it appropriate.

Statistics:

For information on statistics, please see 'Any other information on materials and methods incl. tables'.

Indices:

Maternal Indices:

1) Pregnancy Rate (%): (No. of pregnant females / No. of mated females) x 100

Litter Indices:

1) Male Fetuses (%): (No. of male fetuses / No. fetuses) x 100

2) Female Fetuses (%): (No. of female fetuses / No. fetuses) x 100

3) Pre-Implantation Loss (%): (No. of corpora lutea – No. of implantations / No. of corpora lutea) x 100

4) Post-Implantation Loss (%): (No. of implantations – No. of live fetuses / No. of implantations) x 100

5) Litter % of Fetuses with Abnormalities: (No. of fetuses in litter with a given finding / No. of fetuses in litter examined) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the surviving animals , the following symptoms were observed: Coloured discharge, (mainly seen on tray below the cage, suggesting coloured urine,) was observed for 2 out of 16 females in the Low dose group, 11 out of 15 females in the Mid dose group and 17 out of 17 females in the High dose group intermittently between GDs 10 and 28. Samples were taken from the urine in the cage tray for a few representative High dose females - the urine collected from the trays was centrifuged and the sediment examined microscopically, it is noted that after centrifugation the supernatant was clear. Small red particles were observed in the urine sediment, the red unknown particles were confirmed not to be red blood cells.

Thin appearance was observed for two out of 16 animals in the High dose group from GD 24 to GD 28. Slightly decreased activity was observed for two out of 16 animals in the High dose group from GD 23 to GD 27. The symptoms above were considered as test item related effects. The following clinical signs were noted during the study but were considered not to be test item related as the incidence was similar across the groups, including the Controls: liquid/soft/few faeces, alopecia, scab.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One Low dose female was found dead on GD17. One female in the Control group, two Low dose females and four females in each of the Mid and High dose groups aborted between GDs 24 and 28 and were killed prematurely. There was a statistically significant difference indicating a trend in increased incidence of abortion between the Controls and the does in the Mid and High dose groups. One Mid dose and one High dose female were killed prematurely on GD 23 and 24, respectively, due to body weight loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item-related effects were observed in the body weight and body weight gain for does in the High dose group during the treatment period. There was a slight, but biologically significant effect on the body weight of the does receiving the High dose during the treatment period (-5.2%), reaching statistical significance on Day 27 (p<0.05). The body weight of the does receiving the Low dose was also decreased when compared to the control during the study without statistical significance. This decrease was considered as normal.

A lower body weight gain was observed in does in the High dose group during the treatment period (-46.1%) so that overall body weight gain for the treatment period was statistically significantly (p<0.01) lower than Controls. There was a similar tendency observed in the Low dose group with a smaller effect (- 10.1%) and without statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item related effect on food consumption in any of the dosed groups.
Statistically significantly decreased mean food consumption was observed between GDs 12-15 in does in the Low (p>0.01, by -35.2%) and in the Mid dose (p>0.05, by -26.7%) groups, when compared with Controls. Statistically significantly decreased mean food consumption was observed between GDs 15-18 in does in the High dose group (p>0.01, by -32.1%). Decreased mean food consumption was observed through the whole study (between GDs 6-27) in all dosed groups without significance or a dose response.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no significant difference in the mean weight of the thyroid glands of treated animals compared to Controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic findings of does at scheduled termination were generally observed with a low incidence without the suggestion of test item-related effects. Changes seen in the liver, kidney and lungs of Control or treated animals were similar to those observed in unscheduled deaths including pale discoloration (liver), depressed area (kidney), discoloration/red focus (lungs). Red multifocal discoloration of the stomach wall, red/white focus in the stomach, mass (soft/firm) in the thoracic cavity were also seen in pregnant terminal Control and/or treated females. All changes were considered to be incidental or a common background finding.

Neuropathological findings:

not examined

Description (incidence and severity):

There was no evidence of any test item-related microscopic changes in examined thyroid and parathyroid glands in does receiving up to a dose level of 1000 mg/kg bw/day test item. A common background finding of unilateral minimal focal mixed mononuclear infiltrate was observed in thyroids of 1/22, 1/22 and 2/22 females from the Control, Low and High dose groups, respectively. Unilateral minimal multifocal congestion of thyroid gland correlated with gross changes was seen in 1/22 females receiving the Mid dose.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One female in the Control group, two Low dose females and four females in each of the Mid and High dose groups aborted between GDs 24 and 28 and were killed prematurely. There was a statistically significant difference indicating a trend in increased incidence of abortion between the Controls and the does in the Mid and High dose groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the extent of pre- and post-implantation loss in the test item treated animals when compared to the control; values were all in the normal range. Numbers of corpora lutea and implantations were similar between the groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the number of total litter losses by resorption in the test item treated animals when compared to the control; values were similar between the groups and all in the normal range.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the numbers of early and late resorptions in the test item treated animals when compared to the control; values were similar between the groups and all in the normal range.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the number of dead foetuses in the test item treated animals when compared to the control; values were similar between the groups and all in the normal range.

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

One female in the Control group, two Low dose females and four females in each of the Mid and High dose groups aborted between GDs 24 and 28 and were killed prematurely. There was a statistically significant difference indicating a trend in increased incidence of abortion between the Controls and the does in the Mid and High dose groups.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Eighty-eight females (22 each for the Control, the Low, Mid, and High dose group, respectively) were inseminated in the study. There were 4, 3, 2 and 1 females not pregnant in the Control, Low, Mid and High dose groups, respectively.

The number of confirmed pregnant, evaluated does at scheduled necropsy was 17, 16, 15 and 16 in the Control, the Low, Mid and High dose group, respectively.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a higher number of foetuses with retarded body weight in the Low and High dose groups (with statistical significance for total numbers of foetuses (p<0.05)), however there was no effect on the number of affected litters. All the runts were found to be normal, other than their weight. Based on the lack of dose-response and there being a similar number of litters affected in the Control group, this was not considered to be an effect of the test item.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Mean numbers of live offspring were similar between the groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The mean foetal sex ratio was similar between the groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter weights in the treated groups showed no statistical difference from the Controls and mean values were within the historical control range. Mean litter size was similar between the groups.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects of the test item on the incidence of external foetal malformations. One variation (Tongue, protruding) was observed for one foetus from the Low dose group (1/157 foetus). One malformation (Forelimbs, hyperflexion) was observed in one foetus from the High dose group (1/140 foetus). There were no external malformations in the foetuses from the Control, Low and Mid dose groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of the test item on the incidence of skeletal malformations and variations. All of the skeletal malformations and variations corresponded to the current historical control or the concurrent study control data or were considered to be incidental findings without dose response. There were 1/1, 1/1, 2/1 and 1/1 foetuses/litters in the Control, Low, Mid and High dose groups, respectively, with one or more skeletal malformations. There were 3/3, 9/5, 6/5 and 9/5 foetuses/litters in the Control, Low, Mid and High dose groups, respectively, with one or more skeletal variations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no visceral malformations in any group in the study.

There were no effects of the test item on the incidence of foetal visceral variations. There were 5/4, 17/10, 9/8 and 6/3 foetuses/litters in the Control, Low, Mid and High dose groups, respectively, with one or more foetal visceral variations, including gall bladder bilobed or misshapen, accessory lung lobe absent, thymic cord and spleen discoloured/malpositioned/small. All findings corresponded with the current historical control data or the study control data or were isolated occurrences that were considered incidental, ascribed to individual variability and not related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

There was no significant difference between the mean crown / rump length of the litters compared to the control.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryo-fetal development

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

The analysis showed that the mean concentration of all formulations was found to be in the range of 95-108% of their nominal concentrations (66.7, 200, 666.67 mg/mL) and were found to be homogenous. No test item was detected in the vehicle control formulations. Based on these analytical results, test item formulations were considered suitable for the study purposes.

 

Table 1. Summary of the Dose formulation analysis
Date of Sample collection	Nominal concentration (mg/mL)	Mean Measured concentration (mg/mL)	Percentage of the nominal concentration (%)	Relative Standard Deviation (%)
24 January 2022	Control	not detected	-	-
	66.7	67.9 ± 1.22	102	1.7
	200	207 ± 2.5	103	1.2
	666.67	721 ± 13.0	108	1.7
21 February 2022	Control	not detected	-	-
	66.7	63.4 ± 1.04	95	1.6
	200	193 ± 2.6	96	1.3
	666.67	646 ± 7.9	97	1.2

 Notes: Samples collected freshly on dates indicated in Table

 

Table 2. Summary of mortality events during the study
Dose Group (mg/kg bw/day)	Animal Number	Day of Death	Comment
0	1519	GD 28	preterminal euthanasia, aborted
100	2503	GD 26	preterminal euthanasia, aborted
	2507	GD 28	preterminal euthanasia, aborted
	2518	GD 17	found dead
300	3505	GD 28	preterminal euthanasia, aborted
	3507	GD 27	preterminal euthanasia, aborted
	3511	GD 23	preterminal euthanasia
	3519	GD 27	preterminal euthanasia, aborted
	3520	GD 28	preterminal euthanasia, aborted
1000	4508	GD 24	preterminal euthanasia, aborted
	4509	GD 27	preterminal euthanasia, aborted
	4512	GD 24	preterminal euthanasia
	4514	GD 27	preterminal euthanasia, aborted
	4516	GD 24	preterminal euthanasia, aborted

GD: Gestation day

 

Table 3. Summary of clinical symptoms for preterminal does
Clinical symptom	Dose (mg/kg bw/day)				Observation	Maximum Longevity (day)
	0 (No. of animals, of 1)	100 (No. of animals, of 3)	300 (No. of animals, of 5)	1000 (No. of animals, of 5)
Animal appeared to be Thin	1	1	2	2	Day 16 - Day 28	8
Decreased activity, slight or moderate	0	3	2	1	Day 16 - Day 28	2
Liquid or soft faeces	0	2	0	3	Day 16 - Day 28	2
Few or no faeces	1	3	5	5	Day 7 - Day 28	13
Coloured discharge (vulva or floor)	0	1	5	5	Day 10 - Day 28	4
Laboured respiration	0	1	1	0	Day 23 - Day 28	1
Respiratory rate increase	0	1	0	0	On Day 16	1
Ataxia, (moderate), incoordination (moderate), recumbency	0	0	1	0	Day 27 - Day 28	2

 

 

Table 4. Summary of clinical symptoms for terminal does, not considered as test item related
Clinical symptom	Dose (mg/kg bw/day)
	0 (No. of animals 17)	100 (No. of animals 16)	300 (No. of animals 15)	1000 (No. of animals 16)	Observation	Maximum Longevity (day)
Liquid or soft faeces	3	2	1	3	Day 0, 22 and 27 on Day 26 and 28 Day 20 - Day 27	2
Few or no faeces	14	14	16	16	Day 7 - Day 28	8
Alopecia	1	1	0	1	Day 9 - Day 28	7
Crust	1	1	0	0	Day 6 - Day 17 Day 26-28	12

 

 

Table 5. Summary of select body weight parameters
Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated does	17	16	15	16
Body weight on GD6 (g)	3950.5	3906.6	3943.1	3922.0	NS
Body weight on GD18 (g)	4236.6	4167.8	4186.3	4078.8	NS
Body weight on GD27 (g)	4386.5	4298.8	4363.4	4157.3*	DN
Body weight on GD28 (g)	4364.5	4307.1	4393.9	4207.9	NS
Body weight gain GD6-27 (g)	436.1	392.1	420.3	235.3**	DN
Gravid uterus weight (g)	556.6	577.7	588.7	514.6	NS
Corrected body weight on GD28 (g)	3807.8	3729.4	3805.2	3693.4	NS
Corrected body weight gain GD28 (g)	114.5	46.3	110.1	38.1	NS
Net body weight gain GD28 (g)	-142.6	-177.2	-137.9	-228.6	NS

Notes: Body weight data were rounded to one decimal place.

Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.

Net body weight gain refers to the corrected body weight on GD28 minus the body weight on GD6.

DN: Dunnett 2 sided test, *= p<0.05, **= p<0.01

NS: Not significant

 

Table 6. Summary of pregnancy data
Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of inseminated females	22	22	22	22
Pre-terminal death or euthanasia	1	3	5	5
Number of non-pregnant females	4	3	2	1
Number of females with ≤ 5 implantation sites	3	3	1	1
Number of evaluated females on GD28 (Caesarean section)	17	16	15	16

 

 

Table 7. Summary of Intra-uterine Evaluation
Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated dams	17	16	15	16
Mean number of corpora lutea	12.41	14.25	14.00	12.88	NS
Pre-implantation loss, mean	2.59	3.50	3.20	2.88	NS
Pre-implantation loss (%), mean	23.88	24.12	21.43	22.98	NS
Mean number of implantations	9.82	10.75	10.87	10.00	NS
Early embryonic loss, mean	0.35	0.50	0.60	0.69	NS
Early embryonic loss (%), mean	2.92	8.88	7.78	6.42	NS
Late embryonic loss, mean	0.82	0.19	0.40	0.31	NS
Late embryonic loss (%), mean	7.21	1.06	3.00	2.76	NS
Dead foetuses, mean	0.18	0.25	0.47	0.25	NS
Dead foetuses (%), mean	1.05	1.90	3.84	2.15	NS
Post-implantation loss, mean	1.35	0.94	1.47	1.25	NS
Post-implantation loss (%), mean	11.17	11.84	14.61	11.33	NS
Total intra-uterine mortality, mean	3.94	4.44	4.67	4.13	NS
Total intra-uterine mortality (%), mean	33.67	31.00	32.07	31.80	NS
Viable foetuses, mean	8.47	9.81	9.40	8.75	NS

NS: Statistically not significant when compared to the vehicle control

Table 8. Examination of viable foetuses
Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of examined litters	17	16	15	16
Viable foetuses, mean	8.47	9.81	9.40	8.75	NS
Male foetuses, mean	4.41	4.50	4.33	4.06	NS
Female foetuses, mean	4.06	5.31	5.07	4.69	NS
Total number of foetuses	144	157	141	140	NS
Total number of male foetuses	75	72	65	65	NS
Total number of female foetuses	69	85	76	75	NS
Sex distribution (% of males / females)	52/48	46/54	46/54	46/54	NS
Mean foetal weight / litter (g)	40.580	39.521	40.529	37.218	NS
Crown / rump length Data Male & Female (mm)	95.3	94.6	94.9	91.7	NS
Number of foetuses with retarded body weight/litter	5	16*	7	16*	CH
Number of affected litters (with retarded body weight, runts)	4	5	3	5	NS

NS: Statistically not significant when compared to the vehicle control.
CH: Chi square, *: p<0.05.
Runt: a foetus is considered to be a runt (growth-retarded) when their body weight is less than the control average weight – 2 standard deviations)

 

Table 9. Summary of foetal abnormalities
Parameter	Dose (mg/kg bw/day)
	0	100	300	1000
External Abnormalities
Total number of examined litters	17	16	15	16
Total number of examined foetuses	144	157	141	140
Total number of intact (normal) foetuses	144	156	141	139
Total number of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	1 / 1
Total number of foetuses / litters with variation	0 / 0	1 / 1	0 / 0	0 / 0
Visceral Abnormalities
Total number of examined litters	17	16	15	16
Total number of examined foetuses	144	157	141	140
Total number of intact (normal) foetuses	139	140*^CH	132	134
Total number of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	0 / 0
Total number of foetuses / litters with variation	5 / 4	17 / 10*^CH	9 / 8	6 / 3
Skeletal Abnormalities
Total number of examined litters	17	16	15	16
Total number of examined foetuses	144	157	141	140
Total number of intact (normal) foetuses	140	147	133	130
Total number of foetuses / litters with malformation	1 / 1	1 / 1	2 / 1	1 / 1
Total number of foetuses / litters with variation	3 / 3	9 / 5	6 / 5	9 / 5

CH: Chi square, *: p<0.05.

 

Table 10. Details of the foetal visceral abnormalities
Parameter			Dose (mg/kg bw/day)				HC data
			0	100	300	1000
Total number of examined litters			17	16	15	16	187
Total number of examined foetuses			144	157	141	140	1272
Visceral variations
Parameter			Dose (mg/kg bw/day)				HC data
			0	100	300	1000
Gallbladder, Bilobed or Misshapen	Litter incidence	n	1	0	1	0	1
		%	5.9	0.0	6.7	0.0	0.53
	Foetal incidence	n	1	0	1	0	1
		%	0.694	0.000	0.709	0.000	0.08
Lung Accessory Lobe, Absent	Litter incidence	n	0	1	0	0	-
		%	0.0	6.3	0.0	0.0	-
	Foetal incidence	n	0	1	0	0	-
		%	0.000	0.637	0.000	0.000	-
Spleen Discoloured	Litter incidence	n	0	0	0	1	-
		%	0.0	0.0	0.0	6.3	-
	Foetal incidence	n	0	0	0	2	-
		%	0.000	0.000	0.000	1.429	-
Spleen, Malpositioned	Litter incidence	n	0	1	0	0	-
		%	0.0	6.3	0.0	0.0	-
	Foetal incidence	n	0	1	0	0	-
		%	0.000	0.637	0.000	0.000	-
Spleen, Small	Litter incidence	n	0	1	0	0	-
		%	0.0	6.3	0.0	0.0	-
	Foetal incidence	n	0	1	0	0	-
		%	0.000	0.637	0.000	0.000	-
Thymic cord	Litter incidence	n	4	7	7	2	42
		%	23.5	43.8	46.7	12.5	22.46
	Foetal incidence	n	4	14*^CH	8	4	67
		%	2.778	8.917	5.674	2.857	5.27

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful), Not  present in the HC

CH: Chi square, *=p<0.05.

 

Table 11. Details of the foetal skeletal abnormalities
Parameter			Dose (mg/kg bw/day)				HC data
			0	100	300	1000
Total number of examined litters			17	16	15	16	187
Total number of examined foetuses			144	157	141	140	1250
Skeletal malformations
Malformed Vertebrae (Hemivertebrae, Hemicentric, Absent)	Litter incidence	n	0	1	1	0	-
		%	0.0	6.3	6.7	0.0	-
	Foetal incidence	n	0	1	1	0	-
		%	0.000	0.637	0.709	0.000	-
Sternebraes Fused, Split^†	Litter incidence	n	0	0	0	1	4
		%	0.0	0.0	0.0	6.3	2.1
	Foetal incidence	n	0	0	0	1	4
		%	0.000	0..000	0.000	0.714	0.310
Rib (Costal Cartilage) Fused	Litter incidence	n	1	1	1	0	1
		%	5.9	6.3	6.7	0.0	0.53
	Foetal incidence	n	1	1	1	0	1
		%	0.694	0.637	0.709	0.000	0.08
Skeletal variations
Skull: Hyoid Body Unossified	Litter incidence	n	0	2	0	0	-
		%	0.0	12.5	0.0	0.0	-
	Foetal incidence	n	0	2	0	0	-
		%	0.000	1.274	0.000	0.000	-
Sternum: Unossified Sternebra (2 or More)	Litter incidence	n	2	3	4	1	15
		%	11.8	18.8	26.7	6.3	8.02
	Foetal incidence	n	2	4	4	3	15
		%	1.389	2.548	2.837	2.143	1.18
Sternum: Sternum or Sternebra Missaligned	Litter incidence	n	1	0	0	1	7
		%	5.9	0.0	0.0	6.3	3.74
	Foetal incidence	n	1	0	0	1	7
		%	0.694	0.000	0.000	0.714	0.55
Sternum: Sternebra, fused	Litter incidence	n	0	1	0	2	4
		%	0.0	6.3	0.0	12.5	2.1
	Foetal incidence	n	0	1	0	3	4
		%	0.000	0.637	0.000	2.143	0.310
Sternum: Sternebra, Misshapen	Litter incidence	n	1	0	0	0	3
		%	5.9	0.0	0.0	0.0	1.60
	Foetal incidence	n	1	0	0	0	3
		%	0.694	0.000	0.000	0.000	0.24
Ribs: Interrupted	Litter incidence	n	1	0	1	1	1
		%	5.9	0.0	6.7	6.3	0.53
	Foetal incidence	n	1	0	1	2	1
		%	0.694	0.000	0.709	1.429	0.08
Ribs: Fused to Sternum (8th)	Litter incidence	n	1	1	0	0	-
		%	5.9	6.3	0.0	0.0	-
	Foetal incidence	n	1	1	0	0	-
		%	0.694	0.637	0.000	0.000	-
Limbs: Unossified Metatarsal	Litter incidence	n	0	0	0	2	-
		%	0.0	0.0	0.0	12.5	-
	Foetal incidence	n	0	0	0	2	-
		%	0.000	0.000	0.000	1.429	-
Limbs: Pubis Unossified	Litter incidence	n	0	1	1	1	2
		%	0.0	6.3	6.7	6.3	1.07
	Foetal incidence	n	0	1	2	1	3
		%	0.000	0.637	1.418	0.714	0.24

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful), -: Not present in the HC.

 

Applicant's summary and conclusion

Conclusions:

In conclusion, 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DECYL AND OCTYL TRIESTERS (EC: 290-754-9, CAS: 90218-76-1), when administered daily by oral gavage to pregnant New Zealand White rabbits from gestation days GD6 to GD27 at up to 1000 mg/kg bw/day resulted in a small bodyweight effect (5% below control), no effect on clinical signs other than red urine at the Mid and High dose (red unknown particulate matter in the urine). There were no effects on the embryotoxicity or foetotoxicity observed (number of foetuses, body weight, variations) in the study. There were no external or visceral malformations. A skeletal variation (Sternebra, Fused) in 3/16 litters in the High dose group was not statistically significant, it was concluded as a chance event. Some of the effected pups had a retarded body weight in the High dose group but there is no correlation between runts and skeletal malformations / variations. It is concluded that the test item caused no developmental toxicity effects.

The following No-Observed-Adverse-Effect-Levels (NOAEL) were derived:

NOAEL maternal toxicity: 300 mg/kg bw/day
Based on body weight, body weight gain and food consumption observed at 1000 mg/kg bw/day. Red unknown particles in the urine were also observed in the Mid and High dose groups.
NOAEL embryotoxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related intra-uterine effect in any treatment group.
NOAEL foetaltoxicity: 1000 mg/kg bw/day
Based on no foetal growth effects and no treatment related rate of runts.
NOAEL teratogenicity: 1000 mg/kg bw/day
Based on no significant findings and no severe malformations in the study.

Executive summary:

This guideline developmental toxicity study (OECD 414) was performed to assess the effects of the test item, 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DECYL AND OCTYL TRIESTERS (EC: 290-754-9, CAS: 90218-76-1), on embryonic and foetal development, including the organogenesis period, of New Zealand White rabbits in their first pregnancy. The does (one control and three test item treated groups) were treated daily by oral gavage administration, from gestation day 6 (GD 6) up to and including gestation day 27 (GD 27), where the day of insemination was counted as Day 0 of pregnancy (GD 0). Control does were treated with the vehicle (corn oil) only. Caesarean sections, necropsy of does and examination of uterine contents were performed on GD 28.

The dose levels were set by the Sponsor based on the available data and information from previous experimental work, including the results of an Oral (Gavage) Dose Range Finding Toxicity Study in Pregnant New Zealand White Rabbits.

Based on the results from the Dose Range Finding study, doses of 1000, 300 and 100 mg/kg bw/day were selected for the main study and designated High, Mid and Low dose, respectively. The aim was to use the highest dose of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering, and to determine the NOAEL for the test material in this study.

Test item formulations were analysed for concentration twice during the treatment period using a validated GC-FID method. Simultaneously, vehicle control formulations were also analysed for the test item.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

The number of confirmed pregnant, evaluated does at scheduled necropsy was 17/22 in the Control, 16/22 in the Low and High dose groups and 15/22 in the Mid dose group.

Results

All test item formulations were within the range of 95-108% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

One female from the Control group, 2 from the Low dose group, 4 from the Mid dose group and 4 from the High dose group were pre-terminally euthanised between GD 24 and GD 28 following early abortion of their foetuses. Although there was no evidence of a specific toxic effect of the test item, it is likely that treatment was a factor which could have contributed to the higher abortion rate in treated does compared to Controls (possibly stress related). One female from the Mid dose and one from the High dose group were preterminally euthanised due to weight loss. The Low, Mid and High dose group females showed a red colour discharge on the tray under the cage (was in the urine; in the absence of evident kidney damage). The urine collected from the trays was centrifuged and the sediment examined microscopically. The red suspended particles were confirmed not to be red blood cells.

There was a a slight (~5% below control mean), but biologically significant effect on the body weight and on the food consumption (~32% below control mean) of the does receiving the High dose during the treatment period with no faeces as s clinical observation caused by the test item.

There were no treatment related findings at necropsy. There was no evidence of any test item-related microscopic changes in the thyroid and parathyroid glands from rabbits receiving any dose of the test material.

There were no statistically significant differences in the intra-uterine parameters (number of implantation, corpora lutea, early and late embryonic loss, post implantation loss, total intrauterine mortality and dead foetuses) in the test item treated animals when compared to the controls.

There was no significant difference in the sex distribution of foetuses between the control and treatment groups. The number of foetuses with retarded body weight per litter was unaffected by treatment. There was no statistical difference in the number of runts between the control and treated groups.

There were no significant external, visceral or skeletal malformations in the study. Skeletal examination showed an apparent increased incidence of the variation ‘Sternebra, Fused' in the High dose group (3/16 litters) which was not statistically significant on a litter basis hence it was considered as a chance incidence. There was insufficient maternal toxicity to explain the malformation, based on the traditional parameters of body weight, clinical signs etc. but the presence of red particles in the urine in the Mid and High dose groups may be indicative of a metabolic overload or stress.

In conclusion, 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DECYL AND OCTYL TRIESTERS (EC: 290-754-9, CAS: 90218-76-1), when administered daily by oral gavage to pregnant New Zealand White rabbits from gestation days GD6 to GD27 at up to 1000 mg/kg bw/day resulted in a small bodyweight effect (5% below control), no effect on clinical signs other than red urine at the Mid and High dose (red unknown particulate matter in the urine). There were no effects on the embryotoxicity or foetotoxicity observed (number of foetuses, body weight, variations) in the study. There were no external or visceral malformations. A skeletal variation (Sternebra, Fused) in 3/16 litters in the High dose group was not statistically significant, it was concluded as a chance event. Some of the effected pups had a retarded body weight in the High dose group but there is no correlation between runts and skeletal malformations / variations.

The following No-Observed-Adverse-Effect-Levels (NOAEL) were derived:

NOAEL maternal toxicity: 300 mg/kg bw/day
Based on body weight, body weight gain and food consumption observed at 1000 mg/kg bw/day. Red unknown particles in the urine were also observed in the Mid and High dose groups.
NOAEL embryotoxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related intra-uterine effect in any treatment group.
NOAEL foetaltoxicity: 1000 mg/kg bw/day
Based on no foetal growth effects and no treatment related rate of runts.
NOAEL teratogenicity: 1000 mg/kg bw/day
Based on no significant findings and no severe malformations in the study.