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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

290-754-9

EC Name:

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

Cas Number:

90218-76-1

Molecular formula:

C33H51O6 to C39H66O6

IUPAC Name:

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

Test material form:

liquid

Remarks:

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Group 1 - Control

Doses / concentrations 2

Dose / conc.:

1 300 ppm

Remarks:

Group 2 - Low dose

Doses / concentrations 3

Dose / conc.:

3 800 ppm

Remarks:

Group 3 - Mid dose

Doses / concentrations 4

Dose / conc.:

13 000 ppm

Remarks:

Group 4 - High dose

Doses / concentrations 5

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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

-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.

Effect levels (P0)

1

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

2

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

3

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

4

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.

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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).

Effect levels (F1)

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

Target system / organ toxicity (F1)

1

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

2

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.