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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (dietary) Reproductive Toxicity NOAEL (Rat) = 13000 ppm, equivalent to achieved dosage of 916 mg/kg/day for males and 1372 mg/kg/day for females (OECD 421).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-MAY-2022 to 02-AUG-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The rat is the preferred rodent species for reproduction toxicity testing. The Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility. An adequate historical database is available at the Test Facility for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
- Weight at study initiation: Males: 394-449 g, females: 221-290 g
- Fasting period before study: Not specified
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation periods, delivery and lactation period, when they were paired* or individually housed (with pups), respectively.
*Note: Males were individually housed after their mating finished until the end of the study.
- Use of restrainers for preventing ingestion (if dermal): N/A
- Diet (e.g ad libitum): During acclimatisation, animals were provided with ssniff® SM R/M 'Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance' diet ad libitum. The diet used for two weeks before the start of dosing and for dietary formulations was ssniff® SM R/M-Z+H 'Complete Feed for Rats and Mice - Maintenance' ad libitum. Diets were provided by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany). Animal food consumption per cage was measured and the mean daily food intake per rat was calculated.
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
The standard content of the diet as provided by the Supplier and a copy of the Certificate of Analysis (batch number: ***, expiry date: ***) was archived with the raw data at Charles River Laboratories Hungary Kft. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of
State Public Health and Medical Officer Service. The quality control results are included in the raw data and archived at Charles River Laboratories Hungary Kft. The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 20.3-25.9℃ (target range: 19-25℃)
- Humidity (%): 29-94% (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: 18-MAY-2022 to 02-AUG-2022

Route of administration:

oral: feed

Vehicle:

other: ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice - Maintenance

Details on exposure:

PREPARATION OF DIETARY FORMULATIONS:
Test item was incorporated into the diet and mixed for up to approximately 12 minutes (approximately 6 minutes for premix preparation, and after addition of water (5%) for an additional 6 minutes for preparation of the complete diets). Water for dampening the diet was used. Following mixing, pellets were prepared by simple compression; no binding agents, steam, external heat, any other process or substance was used that might have affected the test item or the quality of the diets. Similar diet preparation procedures were used to generate control diet (0 mg test item /kg diet). The pelleting process was considered not to induce an "unmixing" of the diets or particle segregation and would prevent the potential settling out of the fine-heavy particles that could occur in handling/transport of powder diets.
The prepared diets were stored at room temperature in plastic bags pending and during transport to Charles River Laboratories Hungary Kft. At Charles River Laboratories Hungary Kft., the prepared diets were stored at room temperature based on validation results (5-month stability at room temperature).

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was selected to be administered in the diet as it is one of the possible routes of human exposure.
- Concentration in the diet: 0, 1300, 3800, or 13000/15000* ppm for the control, low dose, mid dose, and high dose groups, respectively. *For the High dose males only, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg/day.
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): Lot number: 4492318 / 4562418 / 4602418 / 4662418 / 4702418
- Purity: N/A

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation occurred (the mating period lasted for 4 days)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Females remained with the same male until copulation occurred
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item concentration and/or homogeneity in the diets was performed, within the known stability period, using extraction from diet and a validated GC-FID method (Gas Chromatography with flame ionization detection) at the Analytical Department of the Test Facility (Study code: 21/019-316ANA). Test item-containing diet samples of an appropriate weight were collected from the diet of each dose group before the start of treatment with the batch and additionally near the end of the use of each batch of diets to determine concentration and homogeneity. Samples were also taken from the control diet for concentration analysis on each occasion. Sampling for the pre-treatment measurements (before the start of use) and during the treatment phase:
Representative diet samples (at least 5 g/sample) were collected at the Test Facility from five different places in a diet container from each dose group, and additionally one sample from the middle of the diet container was collected for the control diet. The first analysis for each dose group (including the control diet) was performed prior to the start of treatment to confirm the dietary concentrations (this was performed for each batch).
Diet samples were kept at room temperature until measurements. Any sample not required for analysis was discarded following acceptance of the results of the formulation analysis by the Study Director.

Acceptance criteria of the concentration analysis was set according to the analytical method validation and the quantified recovery rate, expected to be at 100 ± 20 % of the mean nominal concentration. Acceptance criteria of the homogeneity was that the coefficient of variation (CV) of replicates (five different places of a diet container) should be less than 20 %.

Duration of treatment / exposure:

Dosing of both sexes began after a 2 week pre-exposure period. Males were dosed for at least 28 days (14 days pre-mating and at least 14 days mating/post-mating)
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before necropsy (21-day post-partum dosing).

Frequency of treatment:

Continuously via the diet

Dose / conc.:

0 ppm

Remarks:

Group 1 - Control

Dose / conc.:

1 300 ppm

Remarks:

Group 2 - Low dose

Dose / conc.:

3 800 ppm

Remarks:

Group 3 - Mid dose

Dose / conc.:

13 000 ppm

Remarks:

Group 4 - High dose

Dose / conc.:

15 000 ppm

Remarks:

Group 4- High dose (males), the dose level was increased to 15 000 ppm from Day 20.

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The concentrations of the test item in ssniff® SM R/M-Z+H “Complete Diet for Rats and Mice- Breeding and Maintenance” diet were selected by the Sponsor in consultation with the Study Director, based on the available data and information from a DRF study (21/019-209PE). Based on those results 13000 ppm was selected as the High dose for this study by the Study Monitor. Lower doses were spaced with a factor of ~3, with the aim of achieving dose levels of approximately 100, 300 and 1000 mg/kg/day test item. The study was extended from PND13 to PND21 as it served as a dose range-finder for an upcoming OECD 443 study.

- Rationale for animal assignment (if not random):
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were allocated to dose groups one day prior to the start of the treatment. Any unused, spare animals were moved back to the stock colony approximately 3 weeks after the start of dosing.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality once per day in the pre-treatment period and twice daily in the treatment period (at the beginning and end of each working day). Any clinical sign noted during dosing or at any other occasions were recorded at the time seen. General (routine) clinical observations were made once a day, during the pre-treatment and treatment period in the afternoon (pm).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period and once before the first exposure on Day 0 (to allow for within-subject comparisons), then at least weekly (in the morning (am)) and before necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and 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), difficult or prolonged parturition or 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. Pertinent behavioural changes, signs of difficult or prolonged parturition were recorded including onset, degree and duration of signs as applicable. On Gestation Day 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat). Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed at least weekly during the pre-exposure and treatment periods and at termination. Females were also weighed on gestation Days GD 0, 7, 14 and 20 and on PPD0 (post-partum day 0, i.e. within 24 hours after parturition), 4, 7, 10, 13 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was determined by weighing the non-consumed diet weekly (on a body weight measurements day). For females, food consumption was also measured on Gestation Days (GD) 0, 7, 14 and 20, and on PPD (Post-partum Day) 0, 4, 7, 13 and 21.
Main daily food consumption was calculated for each interval. Food consumption per cage was measured once weekly and the mean weekly food consumption and daily feed intake per rat were calculated. Based on food consumption data, the mean test item intake of each group was calculated for reporting purposes on the basis of mg/kg body weight/day, in addition food conversion efficiency (g/g) was calculated as [weekly body weight gain (g)/weekly food consumption (g)]. Individual data was calculated, the food consumption data was also reported on a cage basis (two animals per cage until mating, after successful mating the animals were housed individually).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No water consumption was measured in the study.

OPHTHALMIC EXAMINATIONS: No ophthalmoscopy was measured in the study.

NEUROLOGICAL ASSESSMENT (Functional Observational Battery/locomotor activity): No special neurological assessment was performed in this study, other than the clinical signs observation.

OBSERVATION OF THE DELIVERY PROCESS:
- Females were allowed to litter and rear their offspring. The delivery process was observed and all observations were recorded. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they formed a nest from the bedding material provided and covered their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

- THYROID HORMONE ANALYSIS:
For thyroid hormone analysis, blood samples were taken by venepuncture into tubes containing no anticoagulant as follows:
- from all pregnant dams on PPD 22 (females)
- from all adult males and non-pregnant females at termination.
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. Timing was documented in the raw data.
Blood samples were kept at room temperature from sampling until centrifugation (within 30 minutes of collection ), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 21℃). The resulting serum was divided into three aliquots (volume target of 150 μL for the first and second aliquot and the remaining amount for the third aliquot) and stored in an ultra-freezer (-80±10℃) until analysis.

Samples for adult males were assessed for T4 levels. The analysis was conducted using a validated ELISA method.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears taken daily during the pre-treatment period. Any females that failed to show a 4-5 day cycle were not included in the study. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating. Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded. Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4, PND 13 and PND 21.
All the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) was also recorded on each day. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded. On PND 4, litters were culled to yield, as nearly as possible, 5 males and 5 females per litter. Pups to be culled within each litter were selected at random. In litters of insufficient size where the number of male or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10. All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0). The anogenital distance was normalized to a measure of pup size (the cube root of body weight) for statistical analysis. The number of nipples/areolae in male pups was recorded on PND 13. One male and one female pup per litter were selected for culling for blood sampling on PND 21. All surviving pups were necropsied on PND 21. Pups were examined for gross lesions.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not specified

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not specified

Postmortem examinations (parental animals):

SACRIFICE
All parental animals were sacrificed under anaesthesia (Euthanimal 40% (400 mg/mL sodium pentobarbital)) by exsanguination.

GROSS NECROPSY
Gross necropsy was performed on each animal. Males were euthanized after 29 days of dose administration and females on PND22. Unmated females were euthanised on Day 40. After exsanguination the external appearance was examined. The cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. The number of implantation sites and of corpora lutea was recorded in the females.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all adult animals were determined: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, levator ani plus bulbocavernosus muscle complex, Cowper’s glands, glans penis, brain, adrenal glands, paired ovaries (wet weight), thyroids with parathyroids. Testes and epididymides were weighed individually. The brain was weighed to allow the provision of organ to brain weight ratios.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. The weighed organs, the vagina, oviducts and all organs showing macroscopic lesions of all adult animals were preserved. The testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as above) in animals from the Control and High dose groups and on any animals found dead during the study in all groups
• all macroscopic findings (abnormalities), except of minor order from all animals
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of all males that failed to sire and all females that failed to deliver healthy pups.
Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on PND4 (1 male and 1 female pup for blood sampling) or PND21. Pups were culled by exsanguination and the anaesthetic product (Euthanimal 40% (400 mg/mL sodium pentobarbital)) was diluted for pups’ euthanasia
- These animals were subjected to postmortem examinations as follows:
Dead pups and pups killed on PND4 and/or PND21 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs.

GROSS NECROPSY
Pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded. All remaining pups were culled on PND21.
ORGAN WEIGHTS
Thyroid glands from one male and one female PND21 pup from each litter were weighed and preserved in 10% buffered formalin solution.

THYROID HORMONE ANALYSIS (PUPS)
For thyroid hormone analysis, blood samples were taken by narcotisation into tubes containing no anticoagulant as follows:
- from up to two pups per litter on PND4
- from at least two pups per litter on PND21
The collected pup blood samples were pooled by litter and blood samples were kept on ice from sampling until centrifugation (about 30 minutes of collection) then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4ºC). The resulting plasma was divided in at least two aliquots (volume target of at least 125 µL for the first aliquot and at least 75 µL for the second aliquot aliquots) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.
Samples for the PND 21 pups were assessed for Total thyroxine (T4) levels. The analysis was conducted using a validated ELISA method.

Statistics:

For information on statistical analysis please see 'Any other information on materials and methods incl. tables'.

Reproductive indices:

Mating and Fertility Indices
1) Male Mating Index (%): (Number of males with confirmed mating / Total Number of males cohabited) x 100

2) Female Mating Index (%): (Number of sperm − positive females / Total Number of females cohabited) x 100

3) Male Fertility Index (%): (Number of males impregnating a female / Total Number of males cohabited) x 100

4) Female Fertility Index (%): (Number of pregnant females / Number of sperm - positive females) x 100

5) Gestation Index (%): (Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

Pups’ Mortality and Sex Ratio Indices
1) Survival Index (%): (Number of live pups (at designated time) / Number of pups born) x 100
Note: Survival index on PND13 and on PND21 was calculated from number of pups after culling on PND4 instead of number of pups born.

2) Pre-implantation mortality (%): (Number of corpora lutea-Number of implantations / Number of corpora lutea) x 100

3) Intrauterine mortality (%): (Number of implantations-Number of liveborns / Number of implantations) x 100

4) Total mortality (%): (Number of implantations-Number of viable pups (PND0/4/13/21) / Number of implantations) x 100

5) Sex ratio % (male/females): (Number of male / female pups (PND0/4/13/21) / Number of pups examined (PND0/4/13/21)) x 100

Pre-implantation mortality and intrauterine mortality were calculated together as pre-natal mortality in relevant tables. The Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the Low and Mid dose groups during the study. Piloerection was observed in 2/12 females in the High dose group for several days between PND10-19. Hunched posture was observed in 4/12 females in the High dose group for a few days between PND7-19. Thin fur (both forelimbs and urogenital area) was observed in 1/12 females in the Mid dose group for several days between PND4-21. A scar (tail, 1-2 cm) was observed in 1/12 Control females for a few days between PND10-19. Thin fur and the scar were considered incidental. The limited observations of hunched posture and piloerection in females in the High dose group were considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female from the Mid dose group (#3505) was found dead during delivery. All the pups were born dead. No clinical sign was recorded prior to death. No test item-related findings were observed at necropsy. Dilatation of uterine body+horn with the presence of a pup seen macroscopically corresponded with luminal uterine dilatation noted by light microscope. A specific cause of death was not determined for this animal.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item related effect was observed on the body weight and body weight gain in males in the High dose group up to Day 28 and females in the High dose group during gestation and lactation. Statistically significant body weight loss (p<0.01, by -3.7%) was observed in the males in the High dose group during the first 7 days, thereafter the growth rate was similar to control, although weights remained below control (statistically significant at most time points (p<0.01, by -7.5% on Day 28). There was no test item related effect observed in the males in the Low and Mid dose groups.
For females in the High dose group, in the first week of treatment, a lower body weight gain was observed. From the start of pregnancy to the study termination the females in the High dose group consistently gained less weight than controls. Overall gestation weight gain was -8.2% below the control value. Statistically significantly decreased body weight was observed between PND0-PND13, and statistically significantly decreased body weight gain was observed during lactation between PND0-4 and PND13-21 (p<0.01). The final body weight of females in the High dose group (PPD21) was 4.6% below controls, which was not significant. In the females in the Low dose group, body weights were affected (on PND7 it was decreased by -7.2%, on PPD21 it was 7.3% below control ), but the effect observed was not considered as treatment related. There was no test item related effect observed in the females in the Mid dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower (p<0.01) food consumption values were recorded in the females in the High dose group during gestation and lactation. This finding was considered to be test item related; no effect was seen in lower dose groups or in males.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significantly (p<0.01) lower food conversion efficiency in males in the High dose group during the treatment period, but this effect was considered as not adverse.

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:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis: No thyroid hormone concentration level differences were observed for the males and all values were normal. No relevant changes were noted in the absolute or relative (to body / brain) thyroid weights of the parental males. No histopathology (microscopic) findings were detected in any adult High dose males.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minimal decrease in number of corpora lutea in the ovary comparing to concurrent control routine sections was observed in 4/12 females in the High dose group.
Under the conditions of this study, ovarian changes observed in few females in the High dose group mentioned above may be related to stress, but individual variability due to trimming/sectioning needs to be considered in these females based on microscopic evaluation. The ovarian weights decrease was seen in parallel with larger adrenal weights only in females in the High dose group. These changes suggest a typical stress-related responses in the females in the High dose group. However, the endpoints useful to confirm general stress were not made in this study design (thymus weight, adrenal and thymus histology). Hence the observations made did not allow to make a definitive clear attribution of these microscopic differences to stress. However, there were no differences in mean numbers of corpora lutea between control and females in the High dose group that were assessed at the end of treatment. All other changes were incidental, or a common background effect.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- Pre-exposure period: Each female selected for the study showed regular cycles (mean cycle length of 3.97 - 4.07 days was observed in all groups) before starting the treatment period.
- Exposure period (pre-mating and mating periods): No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.00, 4.00, 3.96 and 4.00 days in the Control, Low dose, Mid dose and High dose groups, respectively). Prolonged oestrus was recorded in two Control females (#1502 and #1509).

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating index was 100% in all groups (males and females). The fertility index was 100%, 92%, 92% and 100% in the Control, Low, Mid and High dose groups, respectively. The gestation index was 100% in all dose groups.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 4 days of pairing (cohabitation). The mean duration of mating was 2.92, 2.25, 2.83 and 2.50 days in the Control, Low, Mid and High dose groups, respectively.

Gestation and Parturition: There was no effect of treatment noted during the gestation period, parturition or post-partum period in any of the dose groups.
The mean duration of pregnancy was comparable in the Control and test item treated groups. As far as it could be observed during the study, the parturition was normal for all animals, except one female in the Mid dose group (#3505) where prolonged delivery was noted. All the pups were born dead and the dam died soon after the end of the delivery.

Pregnancy and Litter data: There were no statistically significant differences in the number of implantation sites. The number of live born pups was similar in each group. There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group.

-Test Item intake: The test item intake calculated for the entire treatment period (between Days 0 and termination) are presented in Table X. The test item intake values (mg/kg bw/day) were calculated from the weekly individual body weight, weekly mean food intake and nominal diet concentration. The mean achieved dose levels were 84.6, 233.5 and 916.0 mg/kg bw/day for the males and 147.4, 412.5 and 1372.4 mg/kg bw/day for the females in the Low (1300 ppm), Mid (3800 ppm) and High (13000 / 15000 ppm) dose groups, respectively. For the males in all dose groups, the actual achieved test item intake values were lower than the target dose levels. In the case of females, the mean test item intake was above the achieved dose levels for gestation and lactation, but this was related to the higher food consumption during these periods.

Key result

Dose descriptor:

NOAEL

Effect level:

3 800 ppm

Based on:

test mat.

Remarks:

corresponding to 412.5 mg/kg bw/day

Sex:

female

Basis for effect level:

clinical signs

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

3 800 ppm

Based on:

test mat.

Remarks:

corresponding to 233.5 mg/kg bw/day for males and 412.5 mg/kg bw/day for females.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

13 000 ppm

Based on:

test mat.

Remarks:

corresponding to 1372.4 mg/kg bw/day

Sex:

female

Basis for effect level:

other: based on no test item related adverse reproductive effects and normal litter sizes in all groups

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

13 000 ppm

Based on:

test mat.

Remarks:

corresponding to 916.0 mg/kg bw/day

Sex:

male

Basis for effect level:

other: based on no test item related adverse reproductive effects and normal litter sizes in all groups.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs observed in pups were all considered to be incidental. “Kink in tail” was observed for one Control pup from PND0 to PND21. “Small” was observed for one Control pup during the first four days, one Low dose pup on PND0, for one Mid dose pup on PND3 and for 9 pups in the High dose during the first 7 days. “Abnormal skin colour” was observed for two Low dose pups until PND2, 4 Mid dose pups from PND 0 to PND3 and 1 High dose pup from PND0 to PND1. “Scar” was observed in one Low dose pup from PND1 to PND4 and for one Mid dose pup from PND3 to PND7. “Absent tail tip” was observed for one Low dose pup from PND0 to PND2 and for one Mid dose pup on PND20 and PND21. “Cold to touch” was observed for one Mid dose pup on PND0 and for 1 High dose pup on PND0. “Fur thin" was observed for one Mid dose pup from PND9 to PND18. Evidence of suckling was recorded for all live born pups in the study except for #1508/5, #2506/6, #4503/15 and #4504/17.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation). The number of viable pups on PND0, PND4, PND13 and PND21 as well as pup survival indices on PND0, PND4, PND13 and PND21 were comparable to control. A statistically significantly decreased total number of pups born was observed in the Mid dose group, but this was due to the lower number of dams in this group. There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The PND0 weights of pups in the High dose group were similar to Controls, but the growth of these pups was significantly less than the controls (the overall weight gain to PND 21 was less than 41.7% of the control value). The weights of pups in the Mid dose group were not different to controls to PND4, after which they were statistically below those of the controls, but the values for weight and growth of pups from the Mid dose group were fully in line with historic control data (above the mean). Data were within the historical control range, hence the pups from the Mid dose group was considered to be unaffected by treatment. The body weight effect in pups from the High dose was considered to be test item related, which may be related to maternal milk production or other causes, resulting in effects on pup body weight growth.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance.
No relevant statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item effect was observed on nipple retention.
No nipples/areolae were present in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The PND 21 High dose pups’ body weights were about -38% the size of the control pups. The absolute thyroid gland weights were ~21% below control and ~28% above control when adjusted for body weight. Because the pups lost weight, the body weight adjusted organ weight data were more relevant. The body weight related weight of the thyroid gland was within the historical control range, and it is considered that there was no test item effect directly on the thyroid weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were observed in pups up to 13000/15000 ppm test substance in the diet.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Thyroid Hormone analysis:
No thyroid hormone (T4) concentration level differences were observed for the pups, all values were normal (despite the fact that pups from the High dose group were much smaller than controls on PND21).

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 800 ppm

Based on:

test mat.

Remarks:

corresponding to 233.5 mg/kg bw/dy for males and 412.5 mg/kg bw/day for females.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

13 000 ppm

System:

endocrine system

Organ:

adrenal glands

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

13 000 ppm

System:

female reproductive system

Organ:

ovary

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Key result

Reproductive effects observed:

no

Dietary Analysis

The mean concentrations of test item in the diet were in the range of 85 to 101% of nominal concentrations (within the ±20% limit), thus they were considered acceptable for use on the study. Diets were proved to be homogeneous.

 

Table 2. Selected body weight parameters of parental animals
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000/15000 ppm
Males
Body weight on Day 0 (g) NS	422.6	421.8	422.1	421.6
	difference (%)	-0.2	-0.1	-0.2
Body weight on Day 28 (g) DN	508.7	509.0	506.3	470.4**
	difference (%)	0.1	-0.5	-7.5
Body weight gain Day 0-28 (g) DN	86.1	87.2	84.2	48.8**
	difference (%)	1.3	-2.2	-43.3
Females
Body weight on Day 0 NS	249.6	247.0	248.6	246.9
	difference (%)	-1.0	-0.4	-1.1
Body weight on Day 14 DN	278.8	261.0	268.7	257.1*
	difference (%)	-6.4	-3.6	-7.8
Body weight gain Day 0-14 (g) DN	29.2	14.0**	20.1*	10.2**
	difference (%)	-52.0	-31.1	-65.1
Body weight on GD20 (g) DN	435.3	413.8	431.5	401.9*
	difference (%)	-4.9	-0.9	-7.7
Body weight gain GD 0-20 (g) NS	153.1	152.1	158.8	140.5
	difference (%)	-0.6	3.7	-8.2
Body weight on PPD7 (g) DN	371.8	345.0	358.5	314.4**
	difference (%)	-7.2	-3.6	-15.4
Body weight on PPD21 (g) DU	361.8	335.2*	359.6	345.1
	difference (%)	-7.3	-0.6	-4.6
Body weight gain PPD 13-21 (g) DU	-19.5	-20.0	-14.2	15.6**
	difference (%)	2.6	-27.2	-179.9
Body weight gain PPD 0-21 (g) DN	24.8	21.8	26.9	43.4**
	difference (%)	-11.8	8.7	75.4
Body weight gain Day 0-PPD21 (g) D	112.2	88.3**	109.1	98.2
	difference (%)	-21.3	-2.7	-12.5

Data (group mean values; n=10-12) were rounded to one decimal place.     

GD=gestation day, PPD=post partum day

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test; DU: Dunn test; D: Duncan’s multiple range test, NS: Statistically not significant when compared to the control.

&:For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.

 

 

Table 3. Summary of Mean food consumption
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000/15000 ppm
Males
Mean Food consumption Day 0-14 (g/animal/day) DN	28.26	31.02**	29.15	28.05
	difference (%)	9.8	3.1	-0.7
Mean Food consumption Day 0-28 (g/animal/day) DN	29.17	30.76*	28.89	28.16
	difference (%)	5.4	-0.9	-3.5
Females
Mean Food consumption Day 0-14 (g/animal/day) NS	22.14	20.57	20.77	19.88
	difference (%)	-7.1	-6.2	-10.2
Mean Food consumption GD 0-20 (g/animal/day) DN	28.37	26.55	27.58	25.75**
	difference (%)	-6.4	-2.8	-9.2
Mean Food consumption PPD 0-21 (g/animal/day) DN	74.87	72.16	71.53	54.10**
	difference (%)	-3.6	-4.5	-27.7

Notes: Mean values rounded to two decimal places are shown.     

GD=gestation day, PPD=post partum day

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

NS: Statistically not significant

&: For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.

 

 

Table 4. Mean test item intake of parental animals
Test Item Intake (mg/kg bw/day)
Dietary concentration (ppm)	Males (Day 0 - termination)	Females (Day 0 – end of mating)	Females (Gestation)	Females (Lactation) PND0-13	Mean for females
1300	84.6	105.0	108.2	229.1	147.4
3800	233.5	304.8	315.0	617.7	412.5
13000 / 15000	916.0	1033.2	1241.3	1842.7	1372.1

For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.

 

 

Table 5. Summary of Food conversion efficiency
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000/15000 ppm
Males
Food conversion efficiency (Day 7-28) DN	0.10421	0.09346	0.10135	0.07165**
	difference (%)	-10.3	-2.7	-31.2
Females
Food conversion efficiency (Day 7-14) NS	0.07899	0.03851	0.06388	0.05548
	difference (%)	-51.2	-19.1	-29.8
Food conversion efficiency (GD 0-20) NS	0.26896	0.28731	0.28654	0.27243
	difference (%)	6.8	6.5	1.3
Food conversion efficiency (PPD 7-13) NS	0.02171	0.02210	0.03711	0.04680
	difference (%)	1.8	70.9	115.6

GD=gestation Day, PPD=post-partum day.

Statistical significance compared to control: ** = p<0.01.

DN: Dunnett’s test, NS: Statistically not significant when compared to the control.

&:For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.

 

Table 6. Summary of reproductive parameters (Males)
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000/15000 ppm
Number of treated animals	12	12	12	12
Number of males used for mating	12	12	12	12
Number of pre-terminal deaths	0	0	0	0
Number of successful mating	12	12	12	12
Number of infertile animals	0	1	1	0
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	92	92	100

For High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.

 

 

Table 7. Summary of reproductive parameters (Females)
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000 ppm
Number of treated animals	12	12	12	12
Number of females used for mating	12	12	12	12
Number of sperm positive females	12	12	12	12
Number of females with no implantation sites	0	1	1	0
Number of pregnant females	12	11	11	12
Number of preterminal deaths during pregnancy	0	0	1	0
Number of pregnant females with live born(s)	12	11	10	12
Number of pregnant females with stillborn pups	0	0	1	0
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	92	92	100
Female gestation index (%)	100	100	100	100

 

 

 

Table 8. Summary of pregnancy evaluation
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000 ppm
Number of evaluated females	12	12	12	12
Oestrous cycle length (days), mean NS	4.00	4.00	3.96	4.00
Number of pregnant females	12	11	11/10#	12
Duration of pregnancy (days) NS	22.75	22.64	22.40	22.33
Number of corpora lutea, mean  NS	17.08	15.25	16.58	15.83
Number of implantations, mean NS	16.08	14.42	15.75	16.42
Number of pups born, mean NS	14.83	14.73	15.60	15.75
Number of live born pups, mean NS	14.75	14.55	13.82	15.50
Pre-natal mortality, mean NS	1.33	1.18	3.36	0.92
Pre-natal mortality (%), mean NS	9.17	7.81	21.98	5.20
Post-natal mortality, mean PND0-4 NS	0.17	0.09	0.20	0.08
Post-natal mortality (%), mean PND0-4 NS	1.39	0.57	1.32	0.46
Total mortality, mean PND0-4 NS	1.50	1.27	2.80	1.00
Total mortality (%), mean PND0-4 NS	10.42	8.35	15.29	5.64

NS: Statistically not significant when compared to the control.

#: One Mid dose dam died after the delivery with no live born pups.

 

 

Table 9: Summary of pre-natal and post-natal mortality
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000 ppm
Number of evaluated litters	12	11	10	12
Number of pups born, mean NS	14.83	14.73	15.60	15.75
Number of live born pups, mean NS	14.75	14.55	13.82	15.50
Number of living pups on PND21, mean NS	9.58	10.00	10.00	10.00
Pre-natal mortality, mean NS	1.33	1.18	3.36	0.92
Pre-natal mortality (%), mean NS	9.17	7.81	21.98	5.20
Post-natal mortality on PND0-4, mean NS	0.17	0.09	0.20	0.08
Post-natal mortality on PND0-4 (%), mean NS	1.39	0.57	1.32	0.46
Total mortality on PND4, mean NS	1.50	1.27	2.80	1.00
Total mortality on PND4 (%), mean NS	10.42	8.35	15.29	5.64
Post-natal mortality on PND0-21, mean NS	0.17	0.09	0.20	0.08
Post-natal mortality on PND0-21 (%), mean NS	1.39	0.57	1.32	0.46
Total mortality on PND21, mean NS	1.50	1.27	2.80	1.00
Total mortality on PND21 (%), mean NS	10.42	8.35	15.29	5.64
Survival index on PND0 NS	99.51	98.77	97.55	98.61
Sex ratio % (Female) on PND0 NS	50.45	52.67	44.64	44.74
Survival index on PND4 NS	98.61	99.43	98.68	99.54
Sex ratio % (Female) on PND4 NS	49.71	52.33	43.93	44.93
Survival index on PND13 NS	100.00	100.00	100.00	100.00
Sex ratio % (Female) on PND13 NS	48.21	52.73	48.00	49.17
Sex ratio % (Female) on PND21 NS	48.21	52.73	48.00	49.17

NS: Statistically not significant when compared to the control.

 

 

Table 10. Summary of offspring mortality (F1)
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000 ppm
Number of evaluated litters	12	11	10^#	12
Number of pups born CH	178 / 12	162 / 11	156** / 11	189 / 12
Number of cannibalized pups	0 / 0	0 / 0	1 / 1	0 / 0
Number of autolyzed pups PND0	1 / 1	2 / 2	3 / 3	3 / 3
Number of stillborn pups	0 / 0	0 / 0	0 / 0	0 / 0
Number of live born pups NS	177 / 12	160 / 11	152 / 10	186 / 12
Number of found dead pups (born alive)	0 / 0	0 / 0	0 / 0	0 / 0
Number of living pups on PND0 NS	177 / 12	160 / 11	152 / 10	186 / 12
Number of cannibalized pups (PND0-21)	1 / 1	0 / 0	2 / 2	1 / 1
Number of autolyzed pups (PND0-21)	1 / 1	1 / 1	0 / 0	0 / 0
Number of found dead, intact pups (PND0-21)	0 / 0	0 / 0	0 / 0	0 / 0
Total number of pups died (born alive) NS	0 / 12	0 / 11	0 / 10	0 / 12
Culled for blood sampling on PND4	20 / 12	20 / 11	19 / 10	24 / 12
Culled for litter standardization on PND4	40 / 12	29 / 11	31 / 10	41 / 12
Number of viable pups on PND21 NS	115 / 12	110 / 11	100 / 10	120 / 12

Statistical significance compared to control: ** = p<0.01; CH: Chi square test

NS: Statistically not significant when compared to the control.

#: In the case of a Mid dose litter (#3505), all the pups were born dead.

 

 

Table 11. Body Weight Data (Offspring: F1)
Parameters	Dose groups (dietary concentration)				HCD (min-max)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000
Number of evaluated litters	12	11	10^#	12	3653
Mean pup body weight (PND0), g NS	6.668	6.640	6.591	6.277	6.06-6.85
Mean pup body weight (PND4), g DN	11.818	11.523	10.859	9.396**	9.85-11.32
Mean pup body weight gain (PND0-4), g DN	5.145	4.879	4.268	3.116**	3.50-4.72
Mean pup body weight (PND13), g DN	36.657	34.185	32.276**	22.487**	26.62-31.76
Mean pup body weight (PND21)^&, g DN	66.254	62.640	60.489*	40.988**	63.64-67.04
Mean pup body weight gain (PND13- 21)^&, g DN	29.596	28.455	28.213	18.501**	25.38-28.16
Mean pup body weight gain (PND0-21)^&, g DN	59.511	55.993	53.881*	34.683**	57.08-60.31

Data were rounded to three decimal places.

&: From about PND13, the pups ate not only milk but diet also.
#: In the case of a Mid dose litter (#3505), all the pups were born dead.

DN: Dunnett’s test, NS: Statistically not significant when compared to the control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

 

 

Table 12. Anogenital Distance (Offspring: F1)
Parameters	Dose groups (dietary concentration)
	Group 1 0 ppm	Group 2 1300 ppm	Group 3 3800 ppm	Group 4 13000
Male pups
Number of evaluated litters	12	11	10	12
Anogenital distance, litter mean of males (mm) NS	3.58	3.49	3.40	3.39
Minimum / Maximum value, litter mean (mm)	3.2 / 4.1	3.2 / 3.8	2.9 / 3.8	3.1 / 3.7
Female pups
Number of evaluated litters	12	11	10	12
Anogenital distance, litter mean of females (mm) NS	1.77	1.75	1.78	1.69
Minimum / Maximum value, litter mean (mm)	1.5 / 2.0	1.4 / 1.9	1.6 / 2.1	1.4 / 1.9

Group mean values were rounded to one or two decimal places. NS: Statistically not significant when compared to the control.

 

 

Table 13 (A). Organ weight data (Parental Generation: P0) Organ weight Dose groups (dietary concentration) Group 1 0 ppm Group 2 1300 ppm Group 3 3800 ppm Group 4 13000/15000 Males Terminal body weight, g DN 484.1 485.2 482.2 444.8** difference (%) 0.2 -0.4 -8.1 Adrenal glands, absolute (g) NS HC: 0.066-.079 0.0773 0.0704 0.0722 0.0696 difference (%) -8.8 -6.6 -9.9 Adrenal glands, relative to body (%) NS HC: 0.012-0.017 0.0160 0.0145 0.0150 0.0157 difference (%) -9.2 -6.4 -1.8 Adrenal glands, relative to brain (%) NS HC: 2.9-3.59 3.560 3.209 3.309 3.226 difference (%) -9.8 -7.1 -9.4

Data were rounded to one to four decimal places.  Statistical significance compared to control: ** = p<0.01.  DN: Dunnett 2 sided test, NS: Statistically not significant when compared to the control.  For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.

Table 13 (B). Organ weight data (Parental Generation: P0) Organ weight Dose groups (dietary concentration) Group 1 0 ppm Group 2 1300 ppm Group 3 3800 ppm Group 4 13000 Females Terminal body weight, g DN 329.0 299.4** 319.3 295.8** difference (%) -9.0 -2.9 -10.1 Adrenal glands, absolute (g) DN HC: 0.078-0.106 0.0880 0.0897 0.0920 0.1129** difference (%) 2.0 4.5 28.3 Adrenal glands, relative to body (%) DN HC: 0.024-0.030 0.0267 0.0299 0.0288 0.0383** difference (%) 12.0 7.8 43.7 Adrenal glands, relative to brain (%) DN HC: 3.82-5.11 4.355 4.478 4.547 5.822** difference (%) 2.8 4.4 33.7 Ovaries, absolute, g DN HC: 0.109-0.157 0.1463 0.1349 0.1400 0.1149** difference (%) -7.8 -4.3 -21.4 Ovaries, relative to body (%) DN HC:0.033-0.046 0.0444 0.0452 0.0439 0.0386* difference (%) 1.6 -1.3 -13.1 Ovaries, relative to brain (%) DN HC: 5.4-7.7 7.26 6.74 6.93 5.89** difference (%) -7.1 -4.5 -18.8 Data were rounded to one to four decimal places. Statistical significance compared to control: * = p<0.05, ** = p<0.01. DN: Dunnett test, NS: Statistically not significant when compared to the control. Bold letters: data out of the historical control range.     Table 14. Summary of Thyroid Hormone Concentrations Parameters Dose groups (dietary concentration) Group 1 0 ppm Group 2 1300 ppm Group 3 3800 ppm Group 4 13000/15000 Male adults Number of evaluated animals 12 12 12 12 T4 concentration (ng/mL) NS 55.17 58.17 56.83 53.17 HC range:37-91 difference (%) 5.4 3.0 -3.6 Thyroid gland weights (g) NS 0.0247 0.0244 0.0233 0.0242 HC range: 0.019-0.029 difference (%) -1.0 -5.4 -2.0 Thyroid gland / body weight (%) NS 0.00511 0.00503 0.00484 0.00544 HC range: 0.004-0.006 difference (%) -1.7 -5.4 6.4 PND 21 pups Number of evaluated litters 12 11 10 12 T4 concentration (ng/mL) NS 52.50 54.00 58.30 60.33 HC range 30-60: difference (%) 2.9 11.0 14.9 Thyroid gland weights (g) U 0.0095 0.0095 0.0096 0.0075** HC range: 0.0042-0.0064 difference (%) 0.4 1.0 -20.7 Thyroid gland / body weight (%) (x10^-4) DN 1.4572 1.5747 1.6009 1.8721** HC range: 1.448-2.092 difference (%) 8.1 9.9 28.5 Data (group mean values) were rounded to two or four decimal places. Thyroid and parathyroid weights were measured together. Thyroid gland weight for one male and one female pup per litter were determined. Pups blood (serum) was pooled for T4 (thyroxin determination. HC: Historical control. NS: Statistically not significant when compared to the control.Statistical significance compared to control: * = p<0.05, ** = p<0.01. DN: Dunnett test, U: Mann-Whitney U test versus control. &:For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.     Table 15. Summary of Histopathology Data - Terminal Euthanised Animals   Male Female Group 1 (Control– 0 ppm) Group 2 (1300 ppm) Group 3 (3800 ppm) Group 4 (13000/ 15000 ppm) Group 1 (Control – 0 ppm) Group 2 (1300 ppm) Group 3 (3800 ppm) Group 4 (13000 ppm) Number of Animals: 12 12 12 12 12 11 10 12 Number of Completed Animals: 12 1 1 12 12 3 0 12 ADRENAL GLAND                 Examined 0 0 0 0 0 0 0 1 Vacuolation, Cortical, Increased, Diffuse; zona fasciculata; bilateral . . . . . . . 1 mild . . . . . . . 1 COAGULATING GLAND                 Examined 12 1 1 12 . . . . Normal 12 1 1 12 . . . . LUNGS                 Examined 0 0 0 0 0 1 0 0 Hemorrhage, Pulmonary; multifocal . . . . . 1 . . minimal . . . . . 1 . . OVARY                 Examined . . . . 11 0 0 12 Normal . . . . 11 . . 8 Not Examined: No Section . . . . 1 0 0 0 Decreased Number, Absent Corpora Lutea; bilateral . . . . 0 . . 4 minimal . . . . 0 . . 4 OVIDUCT                 Examined . . . . 12 0 0 12 Normal . . . . 12 . . 12 PROSTATE                 Examined 12 1 1 12 . . . . Normal 12 1 1 9 . . . . Inflammation; mixed cell, focal 0 0 0 1 . . . . minimal 0 0 0 1 . . . . Inflammation; mixed cell, multifocal 0 0 0 1 . . . . minimal 0 0 0 1 . . . . Cellular Debris; multifocal 0 0 0 1 . . . . minimal 0 0 0 1 . . . . STOMACH                 Examined 0 0 0 0 0 0 0 1 Normal . . . . . . . 1 THYMUS                 Examined 0 0 0 0 0 0 0 1 Cellularity Decreased, Lymphocyte . . . . . . . 1 mild . . . . . . . 1 TESTIS (RIGHT)                 Examined 12 1 1 12 . . . . Normal 12 1 1 12 . . . . TESTIS (LEFT)                 Examined 12 1 1 12 . . . . Normal 12 1 1 12 . . . . VAGINA                 Examined . . . . 12 0 0 12 Normal . . . . 12 . . 11 Cyst, Nos; single . . . . 0 . . 1 EPIDIDYMIS (RIGHT)                 Examined 12 1 1 12 . . . . Normal 12 1 1 12 . . . . EPIDIDYMIS (LEFT)                 Examined 12 1 1 12 . . . . Normal 12 1 1 12 . . . . SEMINAL VESICLES                 Examined 12 1 1 12 . . . . Normal 12 1 1 12 . . . . UTERINE CERVIX+BODY+HORN                 Examined . . . . 12 2 0 12 Normal . . . . 12 0 . 12 Dilation, Luminal . . . . 0 1 . 0 mild . . . . 0 1 . 0 Inflammation, Granulomatous; unilateral . . . . 0 1 . 0 mild . . . . 0 1 . 0 &:For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.   Table 16. Reproduction Data Summary   Group 1 (Control – 0 ppm) Group 2 (1300 ppm) Group 3 (3800 ppm) Group 4 (13000/ 15000 ppm) Males Number of males used for mating 12 12 12 12 Number of successful mating 12 12 12 12 Number of infertile animals 0 1 1 0 Male mating index 100 100 100 100 Male fertility index 100 92 92 100 Female Number of females used for mating 12 12 12 12 Number of sperm-positive females 12 12 12 12 Number of females with no implantation sites 0 1 1 0 Number of pregnant females 12 11 11 12 Number of pre-terminal death during pregnancy 0 0 1 0 Number of pregnant females with live born 12 11 10 12 Number of pregnant females with no live born 0 0 1 0 Female Mating index (%): (Females mated / Females paired) * 100 100 100 100 100 Female Fertility index (%): (Pregnant females / Females mated) * 100 100 92 92 100 Female Gestation index (%): (Females with living pups on Day 1 / Pregnant females) * 100 100 100 100 100 Mean of Number of pairing days 2.92 2.25 2.83 2.50   12 12 12 12  % deviation from control, NS=not significant &:For the High dose males, the dose level was increased to 15000 ppm from Day 20, because there was no marked toxicity observed and to achieve the limit dose of 1000 mg/kg bw/day.       Table 17. Summary of Pup Clinical Signs Day Numbers Relative to Litter Date Clinical Observation 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Group: 1 Dose Level: 0 ppm Cannibalised       1                                     Kink in Tail 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Pup cull         40                                   Pup Cull+BS         20                                   Small 1 1 1 1 1                                   Autolysed 1       1                                   Terminal Euthanasia                                           115 Group: 2 Dose Level: 1300 ppm Abnormal skin colour 1 2 1                                       Pup cull         29                                   Pup Cull+BS         20                                   Scar   1 1 1 1                                   Small 1                                           Absent 1 1 1                                       Autolysed 2     1                                     Terminal Euthanasia                                           110 Group: 3 Dose Level: 3800 ppm Abnormal skin colour 4 3   1                                     Cannibalised 1 1     1                                   Cold to touch 1                                           Fur thin                   10 10 10 10 10 10 10 10 10 10       Pup cull         31                                   Pup Cull+BS         19                                   Scar       1 1 1 1 1                             Small       1                                     Absent                                         1 1 Autolysed 3                                           Terminal Euthanasia                                           100 Group: 4 Dose Level: 13000 ppm Abnormal skin colour 1 1                                         Cannibalised     1                                       Cold to touch 1                                           Pup cull         41                                   Pup Cull+BS         24                                   Small 1 1 1 2 19 10 10 10                             Autolysed 3                                           Terminal Euthanasia                                           120 BS=Blood sampling

Conclusions:

Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were determined:
The NOAEL for systemic toxicity of the parental generation was considered to be 3800 ppm (corresponding to 233.5 mg/kg bw/day for males and 412.5 mg/kg bw/day for females). (based on effects on body weight, body weight gain, food consumption and clinical signs at 13000/15000 ppm, and potential maternal general stress effects).
The NOAEL for reproductive effects of the parental generation was considered to be 13000 / 15000 ppm (corresponding to 916.0 mg/kg bw/day for males and 1372.4 mg/kg bw/day for females). (based on no test item related adverse reproductive effects and normal litters in all groups).
The NOAEL for pup development was considered to be 3800 ppm (corresponding to 233.5 mg/kg bw/dy for males and 412.5 mg/kg bw/day for females). (based on significantly reduced pup body weight gain with normal pup survival, which may be secondary to maternal effects at the High dose).
A High dose level of around 1000 mg/kg bw/d was recommended for the upcoming OECD 443 study.

Executive summary:

The purpose of this regulatory OECD 421 study was to obtain information on the possible toxic effects of the test item 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (EC: 290-754-9, CAS: 90218-76-1) on reproduction and fertility when administered in the diet to male and female Wistar rats at three dose levels. The study also served as a range-finding study for the OECD 443 study.

This reproductive/developmental toxicity screening test study was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy and parturition and also on the development of the F1 offspring from conception to Day 21 post-partum.

The concentrations of the test item in ssniff^®SM R/M-Z+H “Complete Feed for Rats and Mice- Breeding and Maintenance” were selected by the Sponsor in consultation with the Study Director, based on available data. The test item intake was calculated until PND13 because after that time point the pups also ate the diet, hence the PND21 food consumption data consisted of the food intake of the females and also the pups. Four groups of twelve male and twelve female Wistar rats were given concentrations of 0, 1300, 3800 or 13000 ppm in the diet. For High dose males, the concentration of the diet was increased to 15000 ppm from Day 20 onwards, as there was no marked toxicity observed and in order to achieve the limit dose of 1000 mg/kg bw/day. Dosing of both sexes began after a 2 week pre-exposure period. Males were dosed for at least 28 days (14 days pre-mating and at least 14 days mating/post-mating) and females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before necropsy (21-day post-partum dosing).

 

Parameters measured during the study included twice a day mortality checking, daily and weekly clinical observations, body weight and food consumption. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until postnatal day (PND)21. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs from the adult animals or F1 animals were sampled and preserved in appropriate fixative. The thyroxine (T4) levels in the pups on PND21 and in the parental males were also determined at termination.

For the adult animals, a detailed histological examination was performed on the selected list of retained reproductive organs of all animals/sex in the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups and where abnormalities were observed at necropsy.

RESULTS

Dietary concentrations of 1300, 3800 or 13000 ppm (15000 ppm for males from Day 20), corresponded to achieved dosages of 85, 234 and 916 for males and 147, 413 and 1372 mg/kg bw/day for females.   

Parental effects: There was no mortality during the study. Piloerection and hunched posture were observed in some females receiving the High dose during the lactation period only. Males receiving the High dose showed weight loss (-3.7%) in the first 7 days of treatment but recovered and remained below control (statistically significant at most time points (p<0.01, by -7.5% on Day 28). Females receiving the High dose also showed an initial reduction in growth (statistically significant); their final body weight (PPD21) was 4.6% below controls. Lower food consumption values for females (statistically significant), particularly during lactation, tended to reflect the body weight effects in the High dose group. There was no test item related effect observed in males or females in the Low and Mid dose groups. Higher adrenal and lower ovary weight changes seen in High dose females, may have been indicative of general maternal stress. No clearly adverse test item related macroscopic or microscopic changes were recorded at necropsy or histopathology. There were no differences in T4 hormone levels in parental males that were ascribed to the test item.

Reproductive effects on the parental generation: No test item effect on oestrus cycles of parental females was noted. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery or the post-partum/lactation period until PPD21.

Effects on pup development: There were no adverse effects on the F1 offspring viability at birth, clinical signs or physical development. No test item related macroscopic findings were recorded for F1 pups at necropsy. Statistically significantly decreased mean pup body weight and body weight gain was observed in the High dose group only. The weights of pups in the High dose group on PND21 were approximately one third of that of the controls; there were no other adverse effects in the pups in the High dose group. There were no test item related effects observed on nipple retention or anogenital distance. Although there was a very marked effect on pup weight, the pup thyroid hormone levels were similar in the pups in the High dose group and controls on PND21.

CONCLUSION: Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were determined:
The NOAEL for systemic toxicity of the parental generation was considered to be 3800 ppm (corresponding to 234 mg/kg bw/day for males and 413 mg/kg bw/day for females),  based on effects on body weight, body weight gain, food consumption and clinical signs at 13000/15000 ppm, and potential maternal general stress effects.  
The NOAEL for reproductive effects of the parental generation was considered to be 13000 / 15000 ppm (corresponding to 916 mg/kg bw/day for males and 1372 mg/kg bw/day for females), based on no test item related adverse reproductive effects and normal litters in all groups.
The NOAEL for pup development was considered to be 3800 ppm (corresponding to 234 mg/kg bw/day for males and 413 mg/kg bw/day for females), based on significantly reduced pup body weight gain with normal pup survival, which may be secondary to maternal effects at the High dose.
A High dose level of around 1000 mg/kg bw/d was recommended for the upcoming OECD 443 study.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Quality of whole database:

One guideline study in rats available for assessment - NOAEL for reproductive toxicity was 13000 / 15000 ppm (corresponding to 916 mg/kg bw/day for males and 1372 mg/kg bw/day for females), based on no test item related adverse reproductive effects.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A key OECD 421 guideline study was conducted to evaluate the potential effects of the test material (1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters; CAS# 90218-76-1, EC# 290-754-9) on reproduction and fertility when administered in the diet to male and female Wistar rats at three dose levels. The study also served as a range-finding study for an OECD 443 study. This reproductive/developmental toxicity screening test study was intended to provide initial information on possible effects on male and female reproductive performance, such as gonadal function, mating behaviour, conception, pregnancy and parturition and also on the development of the F1 offspring from conception to Day 21 post-partum.

The concentrations of the test item in ssniff^®SM R/M-Z+H “Complete Feed for Rats and Mice- Breeding and Maintenance” were selected by the Sponsor in consultation with the Study Director, based on available data. The test item intake was calculated until PND13 because after that time point the pups also ate the diet, hence  PND21 food consumption data consisted of the food intake of the females and also the pups. Four groups of twelve male and twelve female Wistar rats were given concentrations of 0, 1300, 3800 or 13000 ppm in the diet. For High dose males, the concentration of the diet was increased to 15000 ppm from Day 20 onwards, as no marked toxicity was observed and in order to achieve the limit dose of 1000 mg/kg bw/day. Dosing of both sexes began after a 2 week pre-exposure period. Males were dosed for at least 28 days (14 days pre-mating and at least 14 days mating/post-mating) and females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before necropsy (21-day post-partum dosing).

 

Parameters measured during the study included twice a day mortality checking, daily and weekly clinical observations and body weight and food consumption. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until postnatal day (PND)21. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs from the adult animals or F1 animals were sampled and preserved in appropriate fixative. The thyroxine (T4) levels in the pups on PND21 and in the parental males were also determined at termination.

For the adult animals, a detailed histological examination was performed on the selected list of retained reproductive organs of all animals/sex in the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups and where abnormalities were observed at necropsy.

Dietary concentrations of 1300, 3800 or 13000 ppm (15000 ppm for males from Day 20), corresponded to achieved dosages of 85, 234 and 916 for males and 147, 413 and 1372 mg/kg bw/day for females.   

Parental effects: There was no mortality during the study. Piloerection and hunched posture were observed in some females receiving the High dose during the lactation period only. Males receiving the High dose showed weight loss (-3.7%) in the first 7 days of treatment but recovered and remained below control (statistically significant at most time points (p<0.01, by -7.5% on Day 28). Females receiving the High dose also showed an initial reduction in growth (statistically significant); their final body weight (PPD21) was 4.6% below controls. Lower food consumption values for females (statistically significant), particularly during lactation, tended to reflect the body weight effects in the High dose group. There was no test item related effect observed in males or females in the Low and Mid dose groups. Higher adrenal and lower ovary weight changes seen in High dose females, may have been indicative of general maternal stress. No clearly adverse test item related macroscopic or microscopic changes were recorded at necropsy or histopathology. There were no differences in T4 hormone levels in parental males that were ascribed to the test item.

Reproductive effects on the parental generation: No test item effect on oestrus cycles of parental females was noted. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery or the post-partum/lactation period until PPD21.

Effects on pup development: There were no adverse effects on the F1 offspring viability at birth, clinical signs or physical development. No test item related macroscopic findings were recorded for F1 pups at necropsy. Statistically significantly decreased mean pup body weight and body weight gain was observed in the High dose group only. The weights of pups in the High dose group on PND21 were approximately one third of that of the controls; there were no other adverse effects in the pups in the High dose group. There were no test item related effects observed on nipple retention or anogenital distance. Although there was a very marked effect on pup weight, the pup thyroid hormone levels were similar in the pups in the High dose group and controls on PND21.

Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were determined:
The NOAEL for systemic toxicity of the parental generation was considered to be 3800 ppm (corresponding to 234 mg/kg bw/day for males and 413 mg/kg bw/day for females),  based on effects on body weight, body weight gain, food consumption and clinical signs at 13000/15000 ppm, and potential maternal general stress effects.  
The NOAEL for reproductive effects of the parental generation was considered to be 13000 / 15000 ppm (corresponding to 916 mg/kg bw/day for males and 1372 mg/kg bw/day for females), based on no test item related adverse reproductive effects and normal litters in all groups.
The NOAEL for pup development was considered to be 3800 ppm (corresponding to 234 mg/kg bw/day for males and 413 mg/kg bw/day for females), based on significantly reduced pup body weight gain with normal pup survival, which may be secondary to maternal effects at the High dose.

Effects on developmental toxicity

Description of key information

Rat (OECD 414): Developmental NOAEL = 1000 mg/kg/day
Rabbit (OECD 414): Developmental NOAEL = 1000 mg/kg/day

A key OECD 414 prenatal developmental toxicity study in rats was conducted to evaluate the effects of the test material on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Liberati, 2010). Groups of time mated Sprague Dawley rats were dosed with 0, 100, 300 or 1000 mg/kg/day 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters. 

Maternal toxicity was observed at 1000 mg/kg/day resulting in a NOAEL for maternal toxicity of 300 mg/kg/day and the NOAEL for  developmental toxicity was established at 1000 mg/kg/day.

A key OECD 414 prenatal developmental toxicity study in rabbits was conducted to evaluate the effects of the test material on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Szalóki, 2023). Groups of time mated New Zealand White rabbits were dosed with 0, 100, 300 or 1000 mg/kg/day 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters.

Maternal toxicity was observed at 1000 mg/kg/day resulting in a NOAEL for maternal toxicity of 300 mg/kg/day and the NOAEL for developmental toxicity was established at 1000 mg/kg/day.

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

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

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 - Control

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2 - Low dose

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3 - Mid dose

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

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

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

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.

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

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.

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

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

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.

 

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November to December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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: female approx. 11 weeks (male 13 weeks)
- Weight at study initiation: 221-260 g female, males at least 307 g
- Fasting period before study: no
- Housing: no more than 5 of one sex to a cage, in clear polycarbonate cages (43 x 27 x 18 cm) with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy) ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: approximately 2 weeks before the start of the treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15 to 25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2009-11-12 To: 2009-12-14

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester will be dissolved/suspended in the vehicle. The formulation will be prepared daily. Concentrations will be calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given
- Concentration in vehicle: 20, 60, 200 mg/ml (formulation analysis: 19-21, 60, 216-217 mg/ml)
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure and the stability at 24 hours at room temperature were verified in RTC Study no. 77280 in the range of 10 - 200 mg/mL to confirm that the method was acceptable.
Samples of the formulations prepared for this study on week 1 and week 2 were analysed to check the homogeneity and concentration.
Chemical analysis were carried out by the Analytical Chemistry Department of RTC. Results of the analyses were within the limits of
acceptance stated in the RTC SOPs for suspensions (90-110% for concentration and CV
<10% for homogeneity).

Details on mating procedure:

The females will be paired with male rats. Females will be paired one to one in the home
cage of the male and left overnight. The day of mating, as judged by the presence of sperm
in the vaginal smear or by the presence of a copulation plug, will be considered as Day 0 of
gestation (or Day 0 post coitum). Full mating records will be maintained.

Duration of treatment / exposure:

days 6 to 19 post coitum

Frequency of treatment:

once daily

Duration of test:

up to day 20 of gestation

Dose / conc.:

100 mg/kg bw/day

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.

Dose / conc.:

300 mg/kg bw/day

Remarks:

Basis:
nominal conc.

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:
nominal conc.

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were in agreement with the Sponsor based on a previous study in rats

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was observed daily, for mortality all animals were checked early in the morning and again in the afternoon, at weeends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during the dosing period an additional observation for signs of reaction to treatment was performed at approx. 1,5-2 h after dosing


BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed an Days 0, 6, 9, 12, 15 and 20 post coitum

FOOD CONSUMPTION: Yes, food consumption was measured on Days 6, 9, 12, 15 and 20 post coitum starting from Day 0 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: necropsy (including examination of the external surface and orifices)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number, sex and weight of all live foetuses, number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing), gross evaluation of placentae, uteri or individual uterine horns without visible implantations were immersed in a 20 % solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation

Fetal examinations:

- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: approx. half per litter
- Skeletal examinations: Yes: approx. half per litter
- Head examinations: No

Statistics:

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

Indices:

Pre-implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of implantations) x 100/no. of corpora lutea
Post-implantation loss was calculated as a percentage from the formula: (no. of implantations - no. of live young) x 100/no. of implantations
Total implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of live young) x 100/no. of corpora lutea
Sex ratios of the foetuses were calculated as the percentage of males per litter
All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the litter percentage of affected litters relative to all litters.

Historical control data:

no historical data were usesd for comparison with test data

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
Mortality and fate of animals: No mortality occurred during the study. A total of 4 females were found not pregnant at necropsy: one in the control and mid-
dose groups and two in the low dose group. Total resorption was detected in two low dose females. In our experience both incidences are considered within a normal variability.
The number of females with live foetuses on gestation Day 20 was 23 in the control and mid-dose groups, 20 in the low dose group and 24 in the high dose
group.

Clinical signs: at 1000 mg/kg/day increase of staining of different regions of the body surface, mainly noted on head areas (14 out of 24 females), soft faeces.

Body weight performance: a statistically significant decrease in body weight (approximately from 4% on gestation Day 12 to 17% on gestation Day 20) and body weight gain (approximately from 34% on gestation Day 9 to 54% on gestation Day 20) was found in females at 1000 mg/kg/day, when compared to controls,
indicating a condition of maternal toxicity.

Food consumption: a statistically significant decrease in food consumption was found at 1000 mg/kg/day, when compared to controls, starting from Day 9
post coitum and reaching a decrease of approximately 37% on Day 20 post coitum

Terminal body weight, uterus weight and absolute weight gain: statistically significant reductions in terminal body weight and gravid uterus weight (of
approximately 16%) and absolute weight gain (of approximately 67%) were evident at 1000 mg/kg/day when compared to controls

Macroscopic observations: at post mortem examination was found staining of different regions of the head in 8 out of 24 females at 1000 mg/kg/day group and hairloss of different regions of the body surface in 4 out of 24 females of the same group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity:

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity:

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Foetal weight: foetal weight and consequently litter weight at 1000 mg/kg/day were statistically significantly lower than control (-12 %)
No relevant differences were seen in the other litter data parameters investigated.

Foetal abnormalities and variants: a marked increase in small foetuses (weight less than 2.70 g) was noted at 1000 mg/kg/day compared with all other groups (2 in the control group, 3 in the low dose group, 5 in the mid-dose group and 86 in the high dose group), this finding could be considered as a consequence of the maternal toxicity noted in this group.
Malformations such as astomia and agnatia were detected in one foetus in the mid-dose group (dam no. 115 foetus no. 10) and were confirmed at skeletal examination of the foetus, the anomaly of forepaw detected in one high dose foetus was confirmed as malformation at skeletal examination, the incidence of these major abnormalities are considered within a normal variability.
Malformations such as absence of skull bones, pubis no ossification and malpositioned bones were detected in two mid-dose foetuses and two high dose foetuses, an increased incidence in incomplete or no ossification of most parts of the skeleton was noted in high dose foetuses compared to control foetuses, most of these changes were considered correlated to the lower foetal weight associated with maternal toxicity noted in this group
malformations such as extremely enlarged ureter associated with extreme pelvic dilatation of kidneys were noted in one mid- and one high dose foetus, extremely enlarged ureter was also detected in two control foetuses, because the incidence of these malformations was low and not dose-related, they were considered to be incidental.

Key result

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Conclusions:

At the dosage of 1000 mg/kg/day, the test item caused a marked maternal toxicity as indicated by the reduction in food consumption, body weight, body weight gain, gravid uterus weight and absolute weight gain. As a consequence of the marked maternal toxicity, at the same dosage, foetal toxicity was present as demonstrated by the reduction in litter weight, foetal weight and delay in the ossification of different parts of the foetal skeleton..
Visceral malformations detected in mid- and high dose groups were considered common findings in foetuses, that usually disappear shortly after birth and since the incidence was low and not dose-related, they did not show clear relationship to treatment and therefore were considered incidental.
Skeletal findings detected in the same groups were considered to be a consequence of the low foetal weight and due to the signs of maternal toxicity observed in the high dose group, rather than to a direct effect of the test item on foetuses.
No signs of maternal and foetal toxicity were noted at the dosages of 100 and 300 mg/kg/day.
On the basis of the results obtained in this study, the dosage of 300 mg/kg/day could be considered the NOAEL for maternal toxicity and the dosage of 1000 mg/kg/day the NOAEL for embryo-foetal effects. No exposure related developmental toxic effects were observed in the study.

Executive summary:

1,2,4 -Benzenetricarboxylic acid, decyl octyl ester was tested in rats to detect the effects on pregnant animals, when the material was administered during the period of organogenesis. The study design was based on OECD Guideline of Testing of Chemicals No. 414 "Prenatal" Developmental Toxicity Study", adopted 22nd January 2001.

 

Mated female Sprague Dawley rats were randomised into 3 test groups and a control group, each containing 24 animals. The animals were dosed by oral gavage over Days 6 -19 of gestation. Dose levels administered were 0, 100, 300 and 1000 mg/kg/day.

 

Animals were regularly monitored during gestation for clinical signs of toxicity and for effects on body weight and food consumption performance. They were killed on Day 20 of gestation. The status of each implantation was recorded and the foetuses were weighed and examined for visceral and skeletal abnormalities, including the state of skeletal ossification.

 

On the basis of the results obtained in this study, the dosage of 300 mg/kg/day could be considered the NOAEL for maternal toxicity and the dosage of 1000 mg/kg/day the NOAEL for embryo-foetal effects. No exposure related developmental toxic effects were observed in the study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

other: Rat and Rabbit

Quality of whole database:

One guideline study in rats and one guideline study in rabbits available for assessment

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A key OECD 414 (Rat) guideline study was conducted to evaluate the potential effects of the test item (1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters) on pregnant animals and the developing conceptus, when administered during the period of organogenesis.

Four groups of 24 mated female Sprague-Dawley rats were given 0, 100, 300 or 1000 mg/kg bw/day 1,2,4 -Benzenetricarboxylic acid, dodecyl and octyl triesters, once daily, from Day 6 to Day 19 of gestation (GD6-19). Animals were administered the test item in corn oil by oral gavage at a constant dose volume of 5 mL/kg body weight. Clinical observations, body weight and food consumption were recorded at regular intervals. Animals were euthanised on GD 20 and a macroscopic necropsy was performed. For each animal, the uterus was removed and weighed and the number of corpora lutea, implantations, resorptions and live and dead foetuses was recorded. The sex and body weight of each foetus was determined. Foetuses were examined for external, visceral and skeletal abnormalities.

At the dosage of 1000 mg/kg/day, the test item caused a marked maternal toxicity as indicated by the reduction in food consumption, body weight, body weight gain, gravid uterus weight and absolute weight gain. As a consequence of the marked maternal toxicity, at the same dosage, foetal toxicity was present as demonstrated by the reduction in litter weight, foetal weight and delay in the ossification of different parts of the foetal skeleton.

Visceral malformations detected in mid- and high dose groups were considered common findings in foetuses, that usually disappear shortly after birth and since the incidence was low and not dose-related, they did not show clear relationship to treatment and therefore were considered incidental.

Skeletal findings detected in the same groups were considered to be a consequence of the low foetal weight and due to the signs of maternal toxicity observed in the high dose group, rather than to a direct effect of the test item on foetuses.
No signs of maternal and foetal toxicity were noted at the dosages of 100 and 300 mg/kg/day.

On the basis of the results obtained in this study, the dosage of 300 mg/kg/day could be considered the NOAEL for maternal toxicity and the dosage of 1000 mg/kg/day the NOAEL for embryo-foetal effects. No exposure related developmental toxic effects were observed in the study.

A key OECD 414 (Rabbit) guideline study was conducted to evaluate the potential effects of the test item (1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters) on pregnant animals and the developing conceptus, when administered during the period of organogenesis.

Four groups of 22 time-mated female New Zealand White rabbits were given 0, 100, 300 or 1000 mg/kg bw/day 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters, once daily, from Day 6 to Day 27 of gestation (GD6-27). Animals were administered the test item in corn oil by oral gavage at a constant dose volume of 1.5 mL/kg body weight. Clinical observations, body weight and food consumption were recorded at regular intervals. Animals were euthanised on GD 28 and a macroscopic necropsy was performed. For each animal, the uterus was removed and weighed and the number of corpora lutea, implantations, resorptions and live and dead foetuses was recorded. The sex and body weight of each foetus was determined. Foetuses were examined for external, visceral and skeletal abnormalities.

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 rabbits from gestation days GD 6 to GD 27 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.

On the basis of the results obtained in this study, the dosage of 300 mg/kg/day was considered to be the NOAEL for maternal toxicity and the dosage of 1000 mg/kg/day the NOAEL for embryo-foetal effects. No exposure related developmental toxic effects were observed in the study.

Justification for classification or non-classification

The results of the developmental study and reproduction toxicity investigations show that 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters does not does not warrant classification according to Regulation (EC) No 1272/2008.