Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-05 - 2018-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 2016-07-29
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Production lot
- Lot/batch No.of test material: 221273406F
- Expiration date of the lot/batch: 2019-01-19

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected as the most appropriate species being that preferred by the test guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia SpA, Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P): 12 - 14 weeks
- Weight at study initiation: (P) Males: 419 - 487 g; Females: 264 - 319 g
- Fasting period before study: no
- Housing: Pre-mating: up to 5 of one sex/cage. Mating: 1 male/1 female/cage. Post-mating: males group caged; females individually caged
- Diet: commercially available rodent diet (Mucedola 4RF21) ad libitum
- Water: Municipal supply tap water ad libitum
- Acclimation period: 3 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2018-06-05 To: 2018-08-11

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at concentrations of 25 mg/mL, 75 mg/mL and 250 mg/mL.

VEHICLE
- Justification for use and choice of vehicle: The substance is not soluble in water therefore corn oil was used for preparing formulations appropriate for oral administration.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged how?: individually
- Any deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations for concentration and homogeneity was performed twice during the study period.
Samples were taken from different places from each concentration for analysis of concentration and homogeneity. Similar sampling was undertaken from the vehicle control.
Measured concentrations were within the test facility acceptance criteria of 85 - 115% with CV < 10%.
Duration of treatment / exposure:
Males: 2 weeks prior to mating, during pairing, and continuing for a minimum of 28 days (31 days total)
Females: 2 weeks prior to mating, during pairing, continuing through gestation and parturition to 13 days post-partum (at least 51 days total)
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: n/a

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily
- Cage side observations included: mortality and clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly. Females - weekly then on gestation Days 0, 7, 14 and 20, Days 1, 4, 7 and 13 post-partum

FOOD CONSUMPTION INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly intervals

Oestrous cyclicity (parental animals):
Oestrus cyclicity was monitored by vaginal smears in:
- all stock females for at least 2 weeks before allocation in order to exclude females with irregular cycle.
- females allocated to study starting from Day 1 of dosing up to a positive identification of copulation.
- vaginal smears were also be taken from all study females on termination.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/sex/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: yes
- for external and internal abnormalities to establish, where possible, cause of death

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after at least 28 days treatment
- Maternal animals: All surviving animals on Day 14 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ weights: Adrenal glands, Epididymides, Kidneys, Liver, Ovaries, Prostate gland, Seminal vesicles, Spleen, Testes, Thymus, Thyroid, Uterus.
- Tissues fixed and preserved: Abnormalities, Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), CVlitoral gland, Epididymides, Kidneys, Liver, Mammary area (males and females), Ovaries, Oviducts, Parathyroid glands, Pituitary gland, Prostate gland, Sciatic nerve, Seminal vesicles, Spleen, Testes, Thymus (where present), Thyroid gland, Uterus – cervix, Vagina.

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups killed on termination of treatment. After dehydration and embedding in paraffin wax, sections were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed at 14 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: external examination and sex confirmation by inspection of the gonads

Statistics:
For continuous variables such as body weight, food consumption, hormone determination and organ weight 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 histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n iwas more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test.
Reproductive indices:
Copulation Index: No. of pairs with successful copulation/no. of pairs mated X 100
Fertility Index: No of pregnant females/no. of pairs with successful copulation X 100
Pre-coital interval: No. of nights paired prior to detection of mating
Pre-implantation loss: No. of corpora lutea – no. implantation sites/no. of corpora lutea X 100
Pre-natal loss: No. of visible implantation sites – Live litter size/no. of implantation sites X 100

Offspring viability indices:
Post-natal loss: Live litter size at birth – live litter size at Day 4/live litter size at birth X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was noted in treated animals of both sexes dosed at 1000 mg/kg bw/day. This sign appeared in male animals starting generally from the last week of treatment and in females generally from the second part of the gestation period and was observed until the end of the study.
Half of the males treated at 300 mg/kg bw/day exhibited aggressive behaviour during the last days of study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male treated at 100 mg/kg bw/day was found dead on Day 18 of the mating phase (after a total of 31 days of treatment). No clinical signs were observed prior to death. Necropsy examination showed pelvic dilatation of the right kidney and a dark thymus. Microscopically, the most relevant findings were pelvic dilatation of kidneys and minimal tubular atrophy of a few seminiferous tubules in one testis. The pathological picture did not allow the cause of death to be established but, due to the absence of other signs of toxicity/mortality in other treated animals, this death was considered not to be related to the toxicity of the tested material.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption in treated males and females was comparable to the control group during the study.
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
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
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted. Sporadic lesions, which were seen in both control and treated animals, were considered to be an
expression of spontaneous/or incidental pathology, seen in this species and age of animal.

Spermatogenic cycle: Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis: No changes in thyroid hormones were noted in treated parental males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lack of significant treatment related effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Small pups were the main clinical signs noted in both control and treated animals. No toxicological significance was attributed to these findings which are commonly observed in the litters during the lactation period.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Found dead and/or missing pups were observed both in control and treated groups, with similar incidence.
Body weight and weight changes:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were observed in the weight of thyroid of treated pups, when compared to controls.
Gross pathological findings:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone analysis: Triiodothyronine was increased in two male and two female pups from 4 dams treated at 1000 mg/kg bw/day. Due to the minimal incidence and the absence of changes of the other hormones, this finding was considered to be incidental.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No treatment-related nor significant differences in total litter size, live litter size, mean pup loss (percentage), mean pup weights (percentage) and sex ratio of pups were observed among the treated and the control dams at birth and on Days 1, 4 and 13 post partum.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lack of significant treatment related effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOEL (No Observed Effect Level) for general systemic toxicity, fertility and reproduction parameters was 1000 mg/kg bw/day for males and females.
Executive summary:

The toxic effects of trimethyl benzene-1,2,4-tricarboxylate, as well as any effects item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring have been investigated in Sprague Dawley rats after repeated oral exposure. Doses of 0, 100, 300 and 1000 mg/kg bw/day were administered and males were treated for 14 days prior to pairing, during pairing with females and for a total of 31 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for at least 51 days.

No differences were found in terms of mating performance including the pre-coital interval, copulatory evidence or fertility index. All pregnant females had a comparable length of gestation. Litter data and sex ratios were unaffected by treatment. Similar clinical signs were recorded in control and treated pups during the lactation period. No differences were seen between the control and treated pups in anogenital distance. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. No toxicologically significant changes were observed in organ weights of parental animals and no treatment-related changes were noted at macroscopic and microscopic observations, as well as on the evaluation of the seminiferous tubules respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.

The NOEL (No Observed Effect Level) for general systemic toxicity, fertility and reproduction parameters was therefore considered to be 1000 mg/kg bw/day for males and females.