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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Jul - 07 Oct 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
minor deviations related to source of animals, shipping logistics, freezer warming after all analyses were performed, and purity of ethanol fixation (details were given under material and methods); no influence on study outcome
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
1,2,4-Benzenetricarboxylic acid, tri-C9-11-alkyl esters
EC Number:
304-780-6
EC Name:
1,2,4-Benzenetricarboxylic acid, tri-C9-11-alkyl esters
Cas Number:
94279-36-4
Molecular formula:
C36H60O6 to C42H72O6
IUPAC Name:
tris(C9-11-alkyl) benzene-1,2,4-tricarboxylate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The animals were ordered from Envigo RMS Srl, San Pietro al Natisone (UD), Italy instead of Charles River Italia S.p.A., Calco (Lecco, Italy).
- Age at animal order: 9 week females; 11 week males
- Weight at animal order: 175 - 225 g females; at least 340 g males
- Fasting period before study: no
- Housing: no more than 5 per cage in clear polysulfone cages before mating and after mating
- Diet: A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: water bottles, ad libitum
- Acclimation period: An acclimatisation period of approximately 2 weeks was allowed before the start of mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 15 Jul - 15 Aug 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle to reach the required concentrations of 25, 75 and 250 mg/mL. The preparations were made up to weekly interval based on the stability data. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): The test item was administered orally by gavage at a dose volume of 4 mL/kg bw. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated previously in the range from 25 - 250 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85 - 115%; precision CV < 10%). A 28 h stability at room temperature and a 10 d stability at room temperature were verified in the range from 25 - 250 mg/mL.
Samples of the formulations prepared in Week 1 and in the last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in the laboratory SOPs for suspensions (85 - 115% for concentration and CV% < 10% for
homogeneity).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 / 1
- Length of cohabitation: Overnight (until a positive identification of mating was made)
- Proof of pregnancy: The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation
Duration of treatment / exposure:
All animals were dosed once a day from Day 3 through Day 19 post coitum.
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 20 post coitum
Doses / concentrationsopen allclose all
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:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected by the Sponsor based on previous non-GLP preliminary study
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for (rat) dam blood sampling: on Day 20 post coitum

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily (animals were checked for mortality early in each working day and also in the afternoon)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION: Yes
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 post coitum
- Organs examined: Uterus and ovaries. From all females the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratios of organ weight to brain weight was calculated for each animal. The thyroid was also examined histopathologically.
All animals were killed by carbon dioxide inhalation on Day 20 post coitum and necropsied.
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); number of intra-uterine deaths; gross evaluation of placentae
Blood sampling:
- Serum: Yes
- Plasma: no
- Volume collected: Approximately 1 mL
- The Principal Investigator was inadvertently not notified by email of the shipping date of the serum samples.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (soft tissue and skeletal examination, respectively)
- Anogenital distance of all live rodent pups: yes
- Foetuses for skeletal examination were fixed in 99% ethanol instead of 95%, as stated in the Study Protocol.
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. The criterion for statistical significance was p < 0.05.
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 foetuses) x 100) / no. of implantations

Total implantation loss was calculated as a percentage from the formula:
((no. of corpora lutea - no. of live foetuses) x 100) / no. of corpora lutea

Sex ratios of the foetuses were calculated as the percentage of males.
All derived values (e.g., means, percentages, ratios) first were 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 mean litter percentage of affected litters.
Historical control data:
Historical control data for T3, T4, and TSH were provided.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Sign of hairloss was observed in one single animal of the low dose group, from Day 10 to 19 of the gestation phase. This finding was associated with a slight decrease in the body weight gain on Day 12 post coitum, fully recovered thereafter.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significantly decrease of the body weight gain was recorded on gestation Day 12 in the high dose group (-26%), when compared to controls. The animals then recovered, therefore this reduction was not considered adverse. No other differences occurred during gestation.
Body weight at termination and the absolute weight gain of females were unaffected by treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A slight but statistically significant decrease in food consumption was observed in the mid and high dose groups on gestation Day 6 (up to -13% compared to the control group). This effect was transient occurrence and was not considered adverse. No other changes were seen between treated and control groups.
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:
effects observed, non-treatment-related
Description (incidence and severity):
TSH was statistically higher than controls in females dosed at 1000 mg/kg bw/day (+32%). Even though a number of these animals showed values slightly outside the range of historical data (12 out of 25), no other related changes were observed (i.e. T3, T4, thyroid weight and/or histopathological changes); the above finding was therefore considered to be unrelated to treatment (see 'Any other information on results incl. tables', Table 1 (results) and Table 2 (historical control values)).
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study:
- thyroid hormones
- anogenital distance
- genital abnormalities
- thyroid weight and histopathology
- gravid uterus weight
- litter size
- litter / pup weight
- number of implantations, corpora lutea
- number of embryonic fetal deaths and viable fetuses
- post- / pre-implantation loss
- presence of anomalies (external, visceral, skeletal)
- sex ratio

For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Gravid uterus weight was unaffected by treatment with the test item.
There were no treatment-related thyroid gland weight changes (absolute and relative to brain weights) in treated females, when compared to controls.
Any organ weight variations were within the range of expected variations in SD rats of the same age and considered incidental and unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic observations in treated females receiving the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic observations in the thyroid gland of females
receiving the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no differences in the number of implantation loss and intrauterine deaths between control and treated groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no differences in total litter losses by resorption between control and treated groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no differences in the number early or late resorptions between control and treated groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no differences in the number of dead fetuses between control and treated groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no differences in the number of implantations between control and treated groups.
Other effects:
not examined

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effect observed up to and including the highest dose tested.
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effect observed up to and including the highest dose tested.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter and foetal weights (both sexes) did not differ between control and treated groups.
Isolated cases of small foetuses (body weight below 2.7 g) were found in control, low and mid-dose groups with comparable incidence.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live foetuses did not differ between control and treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of males respect to females did not differ between control and treated groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter size and weights did not differ between control and treated groups.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No differences in the anogenital distance were seen in either male or females foetuses between all treated and control groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Abnormal shape of the hindlimb was observed in two foetuses from litters receiving the vehicle. In addition, one of those two foetuses showed bent tail.
No other external findings were recorded in all groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No major abnormalities were found. Minor abnormalities or variations occurred in all groups and included for example altered ossification (asymmetrical, incomplete or no ossification) of several bones of the skull, sternebrae, forepaws, thoracic vertebrae, pelvic girdle and short or rudimentary supernumerary ribs (14th). The incidence of the affected litters in treated groups was similar or even lower than observed in the control group or without dose relation. Therefore, these findings were considered unrelated to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No major abnormalities were found. The incidences of foetuses or litters with anomalies or variations did not suggest any test item effect.
Other effects:
not examined

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed up to and including the highest dose tested

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: skull
skeletal: forelimb
skeletal: sternum
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: central nervous system
visceral/soft tissue: male reproductive system
Description (incidence and severity):
The observed findings occurred as isolated or incidental findings without a dose-response relationship or were comparable to the control group. Therefore, they were considered non-treatment related.

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Thyroid hormone determination on Day 20 post coitum – Group mean data





















































































Parameter/units



Group



1



2



3



4



T3 nmol/L


 



N



25



25



25



25



Mean



0.963



0.981



1.000



0.975



SD



0.126



0.070



0.185



0.137



T4 nmol/L



N



25



25



25



25



Mean



24.3



23.0



24.1



23.0



SD



5.8



6.5



7.4



6.4



TSH ng/mL



N



25



25



25



25



Mean



8.50



10.49



10.46



11.22+



SD



2.17



3.49



3.19



3.39



* = mean value of group is significantly different from control at p < 0.05


+ = mean value of group is significantly different from control at p < 0.01


N = number per group


SD = standard deviation



 


Table 2: Historical data for Sprague Dawley rat










































































































































































 



Males



Females



T3 historical data for Sprague Dawley rat in serum with the kit Beckman Coulter – Immunotech s.r.o., reference IM1699



Mean



0.82



0.86



SD



0.22



0.25



Minimum



0.40



0.39



Maximum



1.57



1.61



Median



0.78



0.79



5e Centile



0.54



0.55



95e Centile



1.30



1.35



Number



109



251



Years of reference



2020 - 2021



2020 - 2021



Number of studies considered



10



14



T4 historical data for Sprague Dawley rat in serum with the kit Beckman Coulter – Immunotech s.r.o., reference IM1447



Mean



45



22



SD



7.45



7.63



Minimum



29



13



Maximum



68



54



Median



44



19



5e Centile



35



16



95e Centile



57



38



Number



150



252



Years of reference



2020 - 2021



2020 - 2021



Number of studies considered



14



14



TSH historical data for Sprague Dawley rat in serum with the kit Institute of Isotopes Ltd., reference RK-554



Mean



10.3



7.2



SD



5.45



2.56



Minimum



1.6



2.5



Maximum



43.6



18.9



Median



8.9



6.7



5e Centile



5.1



3.8



95e Centile



18.3



11.2



Number



150



252



Years of reference



2020 - 2021



2020 - 2021



Number of studies considered



14



14


Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the test item had no effect on intrauterine development. The NOAEL was concluded to be >/= 1000 mg/kg bw/day.