Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

A reproduction/ developmental toxicity screening test was performed in 2010 with TBPIN according to OECD TG 421. The following NOAELs were obtained: 50 mg/kg bw/day for maternal toxicity (parent generation) and for offspring generation and for reproductive performance 400 mg/kg bw/day (male) and 50 mg/kg bw/day (female).


A modified study according OECD TG 421 in rats was performed in 2021 with TBPIN, in which the parent (P) generation was dosed 10 weeks prior to mating. For the peroxyesters TBPND (CAS 26748-41-4) and TBPPI (CAS 927-07-1) the same modified design was used in two additional OECD TG 421 studies (2021). The rational was to compare the three peroxyesters regarding effects on reproductive performance after 10 weeks pre-mating exposure and to conclude on the possibility to perform only one subsequent OECD TG 443 study for all three peroxyesters (grouping approach). This 10-weeks pre-mating exposure is recommended in the OECD TG 443 study design. All three substances did not show any adverse effects on the parameters of reproductive performance.


Based on this results, only one study according OECD TG 443 with TBPND was initiated for the peroxyester group. This substance was chosen since ECHA requested for TBPND to include Cohort 3 (immuntoxicity, CCH-D-2114493102-56-01/F). The study results from this OECD TG 443 study will be used in a read-across grouping approach for the peroxyester TBPIN to fulfil ECHAs requests on an EOGRT study (CCH-D-2114493103-54-01/F). This approach was also chosen because of animal welfare reasons. The OECD TG 443 study with TBPND will be completed by June 2022 and a dossier update will be submitted as soon as the study results are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2021-05-19 to 2021-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29th July 2016
Deviations:
yes
Remarks:
10 weeks pre-treatment, no hematology and clinical biochemistry measurements, histopathology only performed for ovaries
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
Details on species / strain selection:
The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and reproduction toxicity studies and well known fertility parameters.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.Cserkesz u. 90. 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 38 - 43 days old
Female animals: 33 - 36 days old
- Weight at study initiation:
Male animals: 159 – 178 g
Female animals: 102 – 131 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Mated females were housed individually
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice, Spezialdiäten GmbH, D-59494 Soest, Germany
- Water, ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
Sunflower oil (Helianthi annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in vehicle in concentrations of 50 mg/mL, 125 mg/mL and 175 mg/mL in the formulation laboratory of the Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility three times during the treatment period.

VEHICLE
- Justification for use and choice of vehicle: The test item is not stable in water. Therefore, sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 50, 125, 175 mg/mL
- Amount of vehicle: 2 mL
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females were cohabited with the same male until copulation occurred (10 days for all pairs)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Sperm positive females were caged individually.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of dosing formulations was between the range of 90 and 108 % of the nominal concentrations. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. TBPPI-75-AL in sunflower oil was stable at room temperature for 24 hours (recovery: 98 % at nominal concentrations of ca. 1 mg/mL and ca. 500 mg/mL, both) and in a refrigerator (5 ± 3 °C) for 3 days (recovery: 105 and 103 % at nominal concentrations of ca. 1 mg/mL and ca. 500 mg/mL, respectively).
Duration of treatment / exposure:
All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 100 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114, 115, 118 or 125 days). Non-pregnant female animals and dam not delivered were administered for 95, 97, 98 or 102 days.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 100, 250 and 350 mg/kg bw/day is based on results obtained in previous studies performed with TBPPI-75-AL (14-Day Oral Gavage Dose Range Finding Study with TBPPI-75-AL in the Rat; Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with TBPPI-75-AL in the Rat).
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after the treatment at approximately the same time
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter and on the day of the necropsy. Parental females were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter, then on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4, 7, 14 and 21 post-partum. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption of parent animals was determined weekly by weighing the given and non-consumed diet by weekly interval with an accuracy of 1 g during the treatment period except mating phase: during the premating period and during post-mating period for male animals; premating period, gestation days 0, 7, 14 and 21, lactation days 0, 7, 14 and 21 for female animals.

WATER CONSUMPTION AND COMPOUND INTAKE: No

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as follows:
- from all dams on lactation/ post-natal day 22;
- from all parent male animals at termination on Day 100;
- non-pregnant female animals and not delivered dam at termination, on Days 95-102;
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears daily before the mating started from each animal for two weeks and from the beginning of the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution and examined with a light microscope after drying.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight, weight of prostate and seminal vesicles with coagulating glands, spermatogenesis, histopathology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, 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

On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as
follows:
- from 2-5 pups per litter on post-natal day 4 (except for litters with 9 or less pups)
- from 3-6 pups per litter on lactation/ post-natal day 22;
Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum FT4 and TSH levels on post-natal days 4 and 22.

OTHER:
One or two male and one or two female pups per litter were selected on post-natal day 21 and were identified by individual numbers written with a marker pen on the tail, were caged (2-3 pups/cage) and were administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight were determined twice weekly and were recorded. The food consumption was determined on post-natal day 22, 29 and 35. All pups were subjected to macroscopic necropsy observation on post-natal day 36.
Postmortem examinations (parental animals):
SACRIFICE
Gross necropsy was performed on each parental animal one day after the last treatment. Animals were euthanized by exsanguination after verification of Isofluran-narcosis and were subjected to gross necropsy as follows:
- Parental male animals: on Day 100;
- Dams: on post-partum days 22, 23 or 24 (on Days 114-125);
- Non-pregnant (but mated) female animals and not delivered dam on Days 95, 97, 98 or 102

GROSS NECROPSY
- After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. Paired organs were weighed together. The thyroid weight was not determined because there were no test item related changes in the hypothalamic-pituitary-thyroid function at the thyroid hormone examinations. Histopathological processing and quantitative examinations of the ovaries were performed in female animals in the control and high dose groups. Follicular enumeration (primordial and small growing follicles – primary, secondary, tertiary follicles, corpora lutea, atresia) was performed in the largest surface section of the ovaries. The fixed ovaries were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not determined
Statistics:
The homogeneity of variance between groups was checked by Bartlett’s
homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Reproductive indices:
See table in "Any other information on materials and methods".
Offspring viability indices:
See table in "Any other information on materials and methods".
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse test item related clinical signs in any group, i.e. parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 250 or 350 mg/kg bw/day at the daily clinical observations.
Salivation and nuzzling up the bedding material were observed in all male and female animals at 250 and 350 mg/kg bw/day (12/12, each) from the second/ third week of the study. Both these signs appeared shortly after the treatment and ceased after short duration. Therefore, these signs were judged to be toxicologically not relevant. There were no clinical signs in male or female animals in the control and 100 mg/kg bw/day groups during the entire observation period. In the male animals, salivation and nuzzling up the bedding material were observed at 250 and 350 mg/kg bw/day with variable occurrence from Day 12 onward up to the termination of the study (Day 99). Similarly, salivation and nuzzling up the bedding material were noted for most of female animals at 250 and 350 mg/kg bw/day during the premating, mating, gestation and lactation periods with variable occurrence. Exophthalmos as individual sign was detected in one dam at 250 mg/kg bw/day from gestation day 2 up to the end of the study (L21).
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality at 100, 250 or 350 mg/kg bw/day
during the course of study (male and female).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was reduced in male animals at 250 and 350 mg/kg bw/day. The difference with respect to the control was less than 10 % at 250 mg/kg bw/day therefore, this slight change was judged to be toxicologically not relevant. The mean body weight was comparable in the male animals in the control and 100 mg/kg bw/day groups on each measuring day from Day 0 up to
and including Day 97. Statistical significance with respect to the control was observed at the lower mean body weight gain of low dose treated male animals between Days 76 and 83. At 250 mg/kg bw/day, statistical significance with respect to the control was detected at the slightly lower mean body weight on Days 56, 76, 83, 90, and 97, as well as at the lower mean body weight gain between Days 24-28, 35-42, 42-49, 49-56 and for the study overall (between Days 0 and 97) in male animals. Compared to their control, statistical significance was detected at the lower mean body weight on Days 28, 35 and from Day 49 up to the termination of the study (Day 97) and at the lower mean body weight gain between Days 28-35, 42-49, 49-56, 56-63, 76-83 and for the study overall in male
animals at 350 mg/kg bw/day. In the female animals at 100 mg/kg bw/day, the mean body weight exceeded the control during the pre-mating, gestation and lactation periods (with statistical significance on Day 14 and from Day 21 onwards by weekly interval during pre-mating period and during the entire gestation and lactation periods). In the female animals at 250 and 350 mg/kg bw/day, the mean body weight and body weight gain were comparable to their control during the entire study except for the slightly lower mean body weight gain at 250 mg/kg bw/day between Day 35 and 42. The statistically significant differences with respect to the control (in male and female animals at 100 mg/kg bw/day and in female animals at 250 mg/kg bw/day) were considered to be indication of the biological variation as similar findings were not detected at the highest dose group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related toxic changes in the mean daily food consumption of male or female animals at 100, 250 or 350 mg/kg bw/day. In accordance with the body weight development, the mean daily food consumption was slightly reduced in male animals at 350 mg/kg bw/day and was elevated in female animals at 100 mg/kg bw/day during the premating and gestation periods. The mean daily food consumption was similar in male animals in the control and test item treated groups at 100 and 250 mg/kg bw/day during the pre-mating and post-mating periods. Statistical significances with respect to the control was observed at the slightly lower mean daily food consumption of male animals at 350 mg/kg bw/day on weeks 1, 6, 8 and 13. However, the mean food consumption was lower than in the control at each measurement day. In the female animals at 100 mg/kg bw/day, statistical significances were detected at the slightly higher mean daily food consumption on week 2 and from week 4 up to the end of gestation period when compared to control. The mean daily food consumption was comparable in the female animals in the control and 250 and 350 mg/kg bw/day groups during the pre-mating, gestation ad lactation periods.
These changes in the mean daily food consumption of male and female animals were considered to be toxicologically not relevant because of the minor degree.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Quantitative examinations of ovaries did not reveal toxicologically relevant differences between the control and 350 mg/kg bw/day group. The mean number of primordial, primary, secondary and tertiary follicle were similar in the control and 350 mg/kg bw/day. Statistical significance with respect to the control was observed at the lower mean number of corpora lutea and follicular atresia, which were probably indicative of biological variation.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The FT4 and TSH levels were not adversely affected in the parental male
animals at any dose levels. The FT4 levels were similar in parental male animals in the control and 100and 250 mg/kg bw/day group and was slightly lowered at 350 mg/kg bw/day. This minor change was considered to be toxicologically not relevant as the TSH level was comparable in the control and high dose groups
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The estrous cycle length was slightly elongated with respect to the control at 250 and 350 mg/kg bw/day. However, this slight change was judged to be toxicologically not relevant. There were no significant differences between the control and test item treated groups in the number or percentage of animals with regular/ irregular cycles, in the mean number of cycles, mean number of days in pro-estrus or estrous or diestrous, in the number or percentage of animals in prolonged diestrous during pre-mating period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 100, 250 or 350 mg/kg bw/day. The copulatory and fertility indices were comparable in male animal in the control and test item administered groups. Statistical significances with respect to the control were detected at the slightly lower mean percentage of dams delivered (gestation index) at 350 mg/kg bw/day because one pregnant female animal did not give birth (not-delivered). This minor difference was considered to be toxicologically not relevant and values met the historical control.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring:
There were no adverse test item related clinical signs in the offspring from post-natal day 0 to 22. Slightly higher percentage of cold pups were observed at 350 mg/kg bw/day (28 %) with respect to the control.The percentage of not suckled pups (no milk in the stomach) was slightly higher (25 %) than in the control group in 250 mg/kg bw/day groups. Alopecia was observed on pups in one litter at 250 mg/kg bw/day (8 %). Some pup was pale (2 % at 100 mg/kg bw/day) or was smaller than others (1 % at 250 mg/kg bw/day). Cold and not suckled pups (no milk in the stomach) were observed with the highest incidence mainly on postnatal day 0 or during the first few post-natal days. These signs were considered to be toxicologically not relevant as the signs were transient and were with low incidence or met the historical control.
F1 generation: There were no clinical signs in male or female animals of F1 generation in the control and 100, 250 or 350 mg/kg bw/day during the entire observation period (from post-natal day 22 up to and including post-natal day 35).
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.The extrauterine mortality was comparable in the control, 100, 250 and 350 mg/kg bw/day on post-natal day 0 and from birth to post-natal day 21. The mean number of live births per litter and number of viable pups per litter were comparable in the control and 100, 250 and 350 mg/kg bw/day groups on post-natal days 0, 4 and 21. There was no significant difference between the control and test item treated (100, 250 and 350 mg/kg bw/day) groups in the survival indices on post-natal day 4.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring:
The body weight development of the offspring was not adversely affected by the test item at 100, 250 and 350 mg/kg bw/day from post-natal day 0 up to postnatal day 21. Higher mean litter weight and higher mean litter weight gain were detected at 100 mg/kg bw/day (on post-natal day 14 and between post-natal days 0-4 and 7-14, respectively). At 250 mg/kg bw/day, statistical significance with respect to the control was observed at the lower mean litter weight on post-natal day 7 and at the lower mean litter weight gain between post-natal days 4 and 7. The mean litter weight and litter weight gain were similar in the control and 350 mg/kg bw/day from post-natal day 0 up to termination. The mean body weight exceeded the control in offspring at 100 mg/kg bw/day from post-natal day 4 up to and including post-natal 21. The mean body weight gain was also higher than in the control in pups at 100 mg/kg bw/day during the observation period reaching statistical significance between post-natal days 0-4, 7-14 and 0-21. At 250 mg/kg bw/day, the mean body weight of offspring was comparable with the control although, the mean body weight gain was slightly lower than in the control between post-natal days 4 and 7. The mean body weight and body weight gain were comparable in the control and 350 mg/kg bw/day from post-natal day 0 up to termination. In accordance with the litter weight and offspring’s mean body weight, the mean body weight of male and female offspring – evaluated by gender – was higher than in the control group at 100 mg/kg bw/day on post-natal day 4. The mean body weight of female offspring was lower with respect to the control at 250 mg/kg bw/day. The mean body weight was similar to the control male and female offspring at 350 mg/kg bw/day (evaluated by gender) on post-natal day 4. The toxicological relevance of the enhanced body weight development of offspring at the low dose is questionable as similar finding was not observed at the higher doses.
F1 generation:
The body weight development of the F1 animals was not adversely affected at 100, 250 or 350 mg/kg bw/day from post-natal day 22 up to post-natal day 35.
Minor changes at 100 mg/kg bw/day (female) and at 250 mg/kg bw/day (male and female) were probably indicative of biological variation as similar findings were not detected at the high dose. The body weight and body weight gain were similar in male animals in the control and 100 mg/kg bw/day administered groups during the 14-day observation period. Statistical significance with respect to the control was detected at the lower mean body weight of F1 male animals at 250 mg/kg bw/day from postnatal day 25 up to termination and at the lower mean body weight gain between post-natal days 22-25 and 22-35. The mean body weight was similar in male animals in the control and 350 mg/kg bw/day groups although, the mean body weight gain of these animals was lowered between post-natal days 22-25, 25-29 and 22-35. In the F1 female animals, statistical significances with respect to the control were observed at the higher mean body weight at 100 mg/kg bw/day during the entire observation period and at the lower mean body weight at 250 mg/kg bw/day on post-natal days 25, 32 and 35. The mean body weight gain was comparable in F1 female animals in the
control and at 100, 250 and 350 mg/kg bw/day between post-natal days 22 and 35.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation: The mean daily food consumption was not affected in the control and test item administered male and female animals of F1 generation during the two weeks observation period. Compared to the control, statistical significance was detected at the lowered mean daily food consumption in F1 male animals at 250 mg/kg bw/day during the two weeks observation. The mean daily food consumption was similar to their control in male animals at 100 and 350 mg/kg bw/day and in female animals at 100, 250 and 350 mg/kg bw/day.
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
Sexual maturation:
no effects observed
Description (incidence and severity):
The sex distribution of male and female pups (mean percentage of pups per litter) was comparable in all groups on post-natal days 0, 4 and 21.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in the anogenital distances at 100, 250 or 350 mg/kg bw/day in male or female offspring. The absolute and normalized anogenital distance were similar in male offspring in the control and test item administered groups. Statistical significances were noted for the slightly shorter mean normalized anogenital distance in female offspring at 100 mg/kg bw/day.
Similar finding was not observed at the higher doses therefore, this difference was considered to be of no toxicological relevance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Nipples/areoles were not seen in any of the examined male offspring in the control or 100, 250 or 350 mg/kg bw/day groups on post-natal day 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring: Test item related macroscopic alterations were not found in stillborn offspring subjected to gross pathological examination on post-natal day 0. There were no macroscopic findings in organs or tissues in stillborn pups in the control (4/4) and in 250 mg/kg bw/day (2/2) groups. Slight autolysis and missing head (due to cannibalism) was noted for stillborn pup (1/1) at 350 mg/kg bw/day).
F1 generation: Macroscopic alterations in connection with the test item were not detected in male or female animals of F1 generation at the necropsy. Species specific findings – pyelectasia and hydrometra - were observed in all groups as follows:
pyelectasia (one or both sides):
male: 3/11 control; 7/12 at 100 mg/kg bw/day; 4/12 at 250 mg/kg bw/day; 5/14 at 350 mg/kg bw/day;
female: 1/11 control; 4/12 at 100 mg/kg bw/day; 2/12 at 250 mg/kg bw/day; 4/14 at 350 mg/kg bw/day; hydrometra (slight, moderate or marked): 3/11 control; 2/12 at 100 mg/kg bw/day; 1/12 at 250 mg/kg bw/day; 2/14 at 350 mg/kg bw/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The FT4 and TSH levels were not adversely affected in PN22 offspring at any dose levels.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of the present study, the test substancewas administered at 100, 250 or 350 mg/kg bw/day oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats as far as investigated in this study. The NOAEL for systemic toxicity of male rats is 250 mg/kg bw/day, the NOAEL for systemic toxicity of female rats is 350 mg/kg bw/day NOAEL for reproductive performance of male/ female rats is 350 mg/kg bw/day and the NOAEL for F1 is 350 mg/kg bw/day.
Executive summary:

A modified OECD 421 study in rats was performed. The purpose of this study was to obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 250 and 350 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 250 and 350 mg/kg bw/day doses corresponding to concentrations of 0, 50 mg/mL, 125 mg/mL and 175 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPPI-75-AL in the dosing formulations varied between the range of 90 % and 108 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 100 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114, 115, 118 or 125 days). Non-pregnant female animals and dam not delivered were administered for 95, 97, 98 or 102 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 - 23 post-partum then were subjected to necropsy on post-partum day 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) from 2-5 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-6 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals and dam not giving birth were sampled for thyroid hormone determination at termination on the day of the necropsy (Days 95, 97, 98 or 102). All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands and thyroid glands of all adult animals were preserved. In addition, organs with macroscopic findings were also preserved for a possible histological examination. A quantitative examination of the ovaries was performed in all female animals in the control and high dose groups. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.


There was no mortality at any dose level (100, 250 or 350 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (100, 250 or 350 mg/kg bw/day) during the entire treatment period. Salivation and nuzzling up the bedding material observed at 250 and 350 mg/kg bw/day were with short duration after the administration of the test item therefore these signs were considered to be toxicologically not relevant.


The body weight development was depressed in male animals at 250 and 350 mg/kg bw/day during the pre-mating, mating and post-mating periods. The difference with respect to the control was lower than 10 % at 250 mg/kg bw/day therefore was judged to be toxicologically not relevant.


The mean daily food consumption was slightly reduced in male animals at 350 mg/kg bw/day in accordance with the body weight changes during the entire treatment period. Estrous cycle The examined parameters of the estrous cycle were not adversely affected in any group (control, 100, 250 and 350 mg/kg bw/day). Delivery data of dams There were no significant or dose related differences between the control and test item treated female animals in the examined parameters of the delivery (100, 250 and 350 mg/kg bw/day).


A test item influence on the examined parameters of reproductive performance was not found at any dose level. Serum thyroid hormones The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.


Necropsy examinations did not reveal specific macroscopic alterations related to the effect of the test item in male or female animals at 100, 250 or 350 mg/kg bw/day.


The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 100, 250 or 350 mg/kg bw/day.


There were no toxicologically relevant differences in the mean number of primordial, primary, secondary, tertiary follicles, atresia or corpora lutea at 350 mg/kg bw/day at the quantitative examination of ovaries.


The offspring’s development was not adversely influenced at any dose level. No adverse effects on mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected. There were no clinical signs, changes in body weight or mean daily food consumption or necropsy findings in F1 generation (male and female) after the 14 days administration of 100, 250 or 350 mg/kg bw/day.


Based on these observations the NOAELs were determined as follows:


NOAEL for systemic toxicity of male rats: 250 mg/kg bw/day


NOAEL for systemic toxicity of female rats: 350 mg/kg bw/day


NOAEL for reproductive performance of male/ female rats: 350 mg/kg bw/day


NOAEL for F1 Offspring (PN 0-21): 350 mg/kg bw/day


NOAEL for F1 generation (PN 22-35): 350 mg/kg bw/day

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
A Read-Across Justification will be submitted in a dossier update as soon as the OECD 443 study results are available (please refer to key study).
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
A Read-Across Justification will be submitted in a dossier update as soon as the OECD 443 study results are available (please refer to key study).
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduction of mean number of births (total, live and alive) a
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2021-06-15 to 2021-06-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29th July 2016
Deviations:
yes
Remarks:
10 weeks pre-treatment, no hematology and clinical biochemistry measurements, histopathology only performed for ovaries
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
Details on species / strain selection:
The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and reproduction toxicity studies and well known fertility parameters.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.Cserkesz u. 90. 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 46- 50 days old
Female animals: 41 - 46 days old
- Weight at study initiation:
Male animals: 200 – 240 g
Female animals: 126 – 156 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Mated females were housed individually
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice, Spezialdiäten GmbH, D-59494 Soest, Germany
- Water, ad libitum, tap water
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
Sunflower oil (Helianthi annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in vehicle in concentrations of 25 mg/mL, 75 mg/mL and 150 mg/mL in the formulation laboratory of the Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility three times during the treatment period.

VEHICLE
- Justification for use and choice of vehicle: The test item is not stable in water. Therefore, sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 25, 75, 150 mg/mL
- Amount of vehicle: 2 mL
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females were cohabited with the same male until copulation occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Sperm positive females were caged individually.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of dosing formulations was between the range of 95 % to 109 % of the nominal concentrations. A sufficient stability and homogeneity in the chosen vehicle have been verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPND proved to be stable room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 ± 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL).
Duration of treatment / exposure:
Parental female animals were dosed for 70 days pre-mating, through up to 1-13 days mating period and throughout pregnancy and up to and including lactation days 21-23 and then were sacrificed and subjected to a detailed necropsy. Overall treatment period was therefore 114-127 days for dams. Non-pregnant female animals were administered for 96 days.
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting was based on results obtained in previous studies performed with TBPND in the rat: 14-Day Oral Gavage Dose Range Finding Study with TBPND in the Rat andCombined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with TBPND in the Rat.
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after the treatment at approximately the same time
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter and on the day of the necropsy. Parental females were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter, then on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4, 7, 14 and 21 post-partum. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption of parent animals was determined weekly by weighing the given and non-consumed diet by weekly interval with an accuracy of 1 g during the treatment period except mating phase: during the premating period and during post-mating period for male animals; premating period, gestation days 0, 7, 14 and 21, lactation days 0, 7, 14 and 21 for female animals.

WATER CONSUMPTION AND COMPOUND INTAKE: No

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as follows:
- from all dams on lactation/ post-natal day 22;
- from all parent male animals at termination on Day 99;
- non-pregnant female animals on Day 96;
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears daily before the mating started from each animal for two weeks and from the beginning of the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution and examined with a light microscope after drying.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight, weight of prostate and seminal vesicles with coagulating glands, spermatogenesis, histopathology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, 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

On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as
follows:
- from 2-7 pups per litter on post-natal day 4 (except for litters with 9 or less pups)
- from 3-8 pups per litter on lactation/ post-natal day 22;
Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum FT4 and TSH levels on post-natal days 4 and 22.

OTHER:
One or two male and one or two female pups per litter were selected on post-natal day 21 and were identified by individual numbers written with a marker pen on the tail, were caged (2-3 pups/cage) and were administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight were determined twice weekly and were recorded. The food consumption was determined on post-natal day 22, 29 and 35. All pups were subjected to macroscopic necropsy observation on post-natal day 36.
Postmortem examinations (parental animals):
SACRIFICE
Gross necropsy was performed on each parental animal one day after the last treatment. Animals were euthanized by exsanguination after verification of Isofluran-narcosis and were subjected to gross necropsy as follows:
- Parental male animals: on Day 99;
- Dams: on post-partum days 22, 23 or 24 (on Days 114-127);
- Non-pregnant (but mated) female animals on Day 96

GROSS NECROPSY
- After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. Paired organs were weighed together. The thyroid weight was not determined because there were no test item related changes in the hypothalamic-pituitary-thyroid function at the thyroid hormone examinations. Histopathological processing and quantitative examinations of the ovaries were performed in female animals in the control and high dose groups. Follicular enumeration (primordial and small growing follicles – primary, secondary, tertiary follicles, corpora lutea, atresia) was performed in the largest surface section of the ovaries. The fixed ovaries were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not determined
Statistics:
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Reproductive indices:
See table in "Any other information on materials and methods".
Offspring viability indices:
See table in "Any other information on materials and methods".
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse clinical signs in any group (50, 150 or 300 mg/kg bw/day). Some male animal at 300 mg/kg bw/day showed salivation shortly after the administration for some minutes. Although the behavior and physical condition of these animals were also normal and therefore, salivation was considered to be not adverse. Salivation was noted for some male animal (7/12) at 300 mg/kg bw/day from Day 32 up to and including 65 or 98. Dermal changes were detected in one male animal at 300 mg/kg bw/day (1/12; scar at the neck region, left side, 0.5-1 cm in diameter, in slight, moderate or marked degree between Days 56 and 97) and in one female animal at 50 mg/kg bw/day (1/11 dam; alopecia on the fore limbs between lactation days 8 and 22). Similarly, reddish discoloration of the hair around eye is frequently observed in non-treated animals of this strain of experimental rats with similar age. This finding was detected in one dam from Day 33 up to and including lactation day 21. Scar, alopecia and reddish discoloration around the eye are common findings occurring also in non-treated animals of this strain of experimental rats with similar age. Therefore, these have no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in control, 50, 150 or 300 mg/kg bw/day groups during the course of study (male and female).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of gestation and lactation periods. The mean body weight gain was also reduced in dams at 300 mg/kg bw/day during the gestation period and during the first few days of lactation period.The mean body weight was comparable in the male animals in the control and all test item administered groups on each measuring day from Day 0 up to and including Day 97. Statistical significance with respect to the control was observed at the higher mean body weight gain of male animals at 150 and 300 mg/kg bw/day between Days 0-3 and at the lower mean body
weight gain of male animals at 300 mg/kg bw/day between Days 90-97. These changes in the body weight gain did not influence the mean body weight values of male animals. In the female animals, the mean body weight was comparable in the control and all dosed groups during the entire pre-mating period in-spite of the lower mean body weight gain at 300 mg/kg bw/day between Days 28-35,
56-63 and 0-69. At 150 mg/kg bw/day, the mean body weight gain exceeded the control between lactation day 7-14 in female animals.During the course of the gestation and lactation periods, statistical significance with respect to the control was observed at the lowered mean body weight and body weight gain of dams at 300 mg/kg bw/day as follows:
- lower mean body weight on gestation days 14 and 21 and lower mean body weight gain between gestation days 7-14, 14-21 and 0-21;
- lower mean body weight on lactation days 0, 4, 7, 14 and lower mean body weight gain between lactation days 0-4 and 7-14;
This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse changes in the mean daily food consumption of male or female animals at 50, 150 or 300 mg/kg bw/day. The mean daily food consumption was comparable in male animals in the control and test item treated groups at 50, 150 and 300 mg/kg bw/day during the pre-mating and post-mating periods except for week 13 (between Days 83-90), where statistical significance with respect to the control was observed at the slightly higher mean daily food consumption of male animals at 300 mg/kg bw/day. In the female animals, statistical significances were detected at the slightly lower mean daily food consumption at 50 and 300 mg/kg bw/day on week 9 of pre-mating period and at 300 mg/kg bw/day between gestation days 14 and 21.
These changes in the mean daily food consumption were considered to be toxicologically not relevant because of the minor degree and transient occurrence.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Quantitative examinations of ovaries did not reveal toxicologically relevant differences between the control and 300 mg/kg bw/day group. The mean number of primordial, primary, secondary and tertiary follicle, corpora lutea were similar in the control and 300 mg/kg bw/day. Statistical significance with respect to the control was observed at the higher mean number of follicular atresia. Follicular atresia is a normal - physiological - process in the ovary to regulate the number of follicles in the developing pool. Increase in follicular atresia can be observed secondary due to different causes. Since the number of primary, secondary and tertiary follicles was not changed compared to the control the increased follicular atresia can be judged as a biological variation.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse changes in the serum thyroid hormone (FT4 and TSH) levels at any dose in the parental male animals. The FT4 levels were slightly lower than in the control group in parental male animals at 150 and 300 mg/kg bw/day groups. The TSH concentration was higher than in the control group in parent male animals at 150 and 300 mg/kg bw/day. These minor changes were considered to be of no toxicological relevance as values met well the historical control in the most animals. The changes in FT4 were considered to be toxicologically not relevant considering the lack of significant relevant changes in TSH values.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The examined parameters of the estrous cycle were not affected by the test item at 50, 150 or 300 mg/kg bw/day. There were no significant differences between the control and test item treated groups in the number or percentage of animals with regular/ irregular cycles, in the mean number or length of cycles, mean number of days in proestrus or estrous or diestrous during pre-mating period. The number or percentage of animals in prolonged diestrous was slightly higher in the control group than in the test item treated groups as an indication of biological variation.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The examined parameters of reproductive performance were not adversely affected by the treatment with the test item in male or female animals at 50, 150 or 300 mg/kg bw/day. Statistical significances with respect to the control were detected at the lower mean percentage of fertile male and female animals (fertility indices) at 50 and 300 mg/kg bw/day as a consequence of infertile mating of one pair (1/12) at 50 mg/kg bw/day and two pairs (2/12) at 300 mg/kg bw/day. The values were within the historical control range. The copulatory index and gestation indices were 100 % in all groups.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduction of mean number of births (total, live and alive)
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring: There were no adverse test item related clinical signs in the offspring from post-natal day 0 to 22. Slightly higher percentage of cold pups were detected at 150 and 300 mg/kg bw/day (16 and 24 %, respectively) with respect to the control. Not suckled pups were observed with the highest incidence at 150 and 300 mg/kg bw/day (35 and 29 %, respectively). Alopecia was observed on the whole body of pups in one litter (6 %) at 50 mg/kg bw/day. Additionally, smaller than normal, pale pups, hemorrhage (on the nose, abdomen), bite mark on the abdomen were noted for some pup. These signs were considered to be toxicologically not relevant as the signs were transient.
F1: There were no clinical signs in male or female animals of F1 generation in the control and 50, 150 or 300 mg/kg bw/day groups during the entire observation period (from post-natal day 22 up to and including postnatal day 35).
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality. The extrauterine mortality was comparable in the control, 50, 150 and 300 mg/kg bw/day on post-natal day 0 and from birth to post-natal day 21.
The survival of offspring were not affected by the test item at 50, 150 or 300 mg/kg bw/day on post-natal days 0, 4 and 21. Statistical significances with respect to the control was detected at 300 mg/kg bw/day at the lower mean number of pups (live and alive) on post-natal day 0 and mean number of pups per litter on post-natal day 4. Although, the extrauterine mortality was low and similar in each group. There was no significant difference between the control and test item treated (50, 150 and 300 mg/kg bw/day) groups in the survival indices on post-natal day 4. The survival index was higher at 300 mg/kg bw/day than in the control group on post-natal day 21 due to the lower number of offspring euthanized on postnatal day 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring: The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 up to postnatal day 21. The mean litter weight, litter weigh gain, body weight and body weight gain were comparable in the control and 50 mg/kg bw/day after birth up to and including post-natal day 21. At 150 mg/kg bw/day, statistical significance with respect to the control was detected at the lower mean litter weight gain between post-natal days 0 and 4 however the mean litter weight was similar to the control. The mean pup weight was lower than in the control at 150 mg/kg bw/day on post-natal
days 4, 7 and 14 as a consequence of the lower mean body weight gain between post-natal days 0-4 and 4-7. The mean litter weight and mean litter weight gain were significantly lower than in the control in offspring at 300 mg/kg bw/day from post-natal day 0 up to and including post-natal day 21 reaching statistical significances in the most cases. Compared to the control, statistically significant difference was observed at the lower mean body weight of pups at 300 mg/kg bw/day on post-natal days 7, 14 and 21 as well as at lower mean body weight gain of pups between postnatal days 4-7 and 7-14, as well as between post-natal days 0-21. Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the slightly lower mean weight of male pups at 50, 150 and 300 mg/kg bw/day and of female pups at 150 mg/kg bw/day on post-natal day 4.
F1: The body weight development of the F1 animals was reduced at 150 and
300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. The body weight and body weight gain were similar in the control and 50 mg/kg bw/day administered male animals during the observation period. In the male animals at 150 mg/kg bw/day, the mean body weight was lower than in the control group during the two weeks observation period with statistical significance on post-natal days 22, 25, 29. The mean body weight gain for these animals was comparable to their control. At 300 mg/kg bw/day, the mean body weight and body weight gain of male animals were significantly lower (up to 17% lower body weight) than in the control reaching statistical significances in the most cases.
The body weight and body weight gain were comparable to the control in
female animals at 50 mg/kg bw/day during the observation period. At 150 mg/kg bw/day, the mean body weight of female animals was lower than in the control group during the two weeks observation period with statistical significance on each measuring days. Statistical significance with respect to the control was detected at the lower mean body weight gain for these animals between post-natal days 25 and 29. In the female animals at 300 mg/kg bw/day, the mean body weight and body weight gain were significantly lower than in the control reaching statistical significances for body weight on each measuring days and for body weight gain between postnatal days 22-25 and 25-29.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1: The mean daily food consumption was lowered at 150 and 300 mg/kg bw/day in F1 male and female animals in accordance with the body weight
changes. The mean daily food consumption was similar in the control and 50 mg/kg bw/day groups (male and female) during the 14-day observation period.
Statistical significance with respect to the control was detected at the lower mean daily food consumption of male animals at 150 mg/kg bw/day between post-natal days 22-29 and at 300 mg/kg bw/day between post-natal days 22-29 and 29-35. In the female animals, lower mean daily food consumption was observed at 150 mg/kg bw/day between post-natal days 29-35 and at 300 mg/kg bw/day between post-natal days 22-29 and 29-35.
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
Sexual maturation:
no effects observed
Description (incidence and severity):
The sex distribution of male and female pups (mean percentage of pups per litter) was comparable in all groups on post-natal days 0, 4 and 21.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in the anogenital distances at 50, 150 or 300 mg/kg bw/day in male or female offspring. Statistical significance was observed at the slightly shorter mean absolute anogenital distance in female pups at 150 and 300 mg/kg bw/day. The mean normalized anogenital distance exceeded the control in male pups at 300 mg/kg bw/day and was below the control in female pups at 300 mg/kg bw/day. These differences were with minor degree and within the range of the historical control data. Therefore, were considered to have no toxicological relevance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Nipples/areoles were not seen in any of the examined male offspring in the control or 50, 150 or 300 mg/kg bw/day groups on post-natal day 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring: Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination (stillborn or dead pups on postnatal day 4). In dead pup in the control group (1/1 male), empty stomach and slightly
autolyzed visceral organs were observed. At 50 mg/kg bw/day, there were no macroscopic findings in one dead pup (1/1). In one stillborn pup at 150 mg/kg bw/day (1/7), malformations – missing orifices of eyes, nose and mouth – were observed. There were no macroscopic findings in organs or tissues in stillborn pups: 1/1 at 50 mg/kg bw/day, 6/7 at 150 mg/kg bw/day and 3/3 at 300 mg/kg
bw/day.
F1: Macroscopic alterations in connection with the test item were not detected in male or female animals of F1 generation at the necropsy. Species specific macroscopic findings – renal changes (male and female) and hydrometra (female) – were observed. In male animals, pale kidneys (1/12 control, 1/11 at 300 mg/kg bw/day) and right side pyelectasia (3/12 at 150 mg/kg bw/day; 1/11 at 300 mg/kg bw/day) were observed. In female animals, slight, moderate or marked hydrometra was detected as follows: 1/11 in control group; 1/11 at 50 mg/kg bw/day; 3/14 at 150 mg/kg bw/day). Additionally, pale kidneys (2/11 at 50 mg/kg bw/day; 1/14 at 150 mg/kg bw/day; 1/10 at 300 mg/kg bw/day) and one or both sided pyelectasia (1/11 at 50 mg/kg bw/day; 2/10 at 300 mg/kg bw/day) were seen. Pyelectasia and hydrometra are common macroscopic findings in experimental rats of this strain with similar age.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The FT4 and TSH levels were not adversely affected in PN22 offspring at any dose levels.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
TBPND caused reduction of mean number of births (total, live and alive) after administration of 300 mg/kg bw/day by oral gavage to Han:WIST rats. 50 or 150 mg/kg bw/day did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female animals. At 300 mg/kg bw/day, the body weight development of dams was reduced during the gestation and lactation period. There were no signs of systemic toxicity at 50, 150 mg/kg bw/day (male and female) or 300 mg/kg bw/day (male). The body weight development of the offspring was depressed between post-natal day 0 and 21 after repeated oral administration of dams at 150 or 300 mg/kg bw/day. The body weight development and food consumption of the F1 animals were reduced at 150 and 300 mg/kg bw/day during the 14-day post-weaning treatment. Based on these observations the NOAEL for systemic toxicity of male/ female rats is 300 mg/kg bw/day, the NOAEL for reproductive performance of male/ female rats is 150 mg/kg bw/day and the NOAEL for F1 Offspring is 50 mg/kg bw/day.
Executive summary:

A modified OECD 421 study in rats was performed. The purpose of this study was to determine the dose levels for the main study (OECD 443) and obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 50, 150 and 300 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 150 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 150 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPND in the dosing formulations varied between the range of 95 % and 109 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 99 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114-127 days). Non-pregnant female animals were administered for 96 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to days 21 post-partum then were subjected to necropsy on post-partum days 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-8 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals were sampled for thyroid hormone determination at termination, on Day 96.


All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, thyroid glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one female animal at 50 mg/kg bw/day, liver, spleen, heart, lungs in one dam at 150 mg/kg bw/day as well as the kidneys of some animal were also preserved for a possible histological examination. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. A quantitative examination of ovaries was performed in all female animals in the control and high dose groups.


There was no test item related mortality at any dose level (50, 150 or 300 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (50, 150 or 300 mg/kg bw/day) during the entire treatment period. Salivation observed in male animals at 300 mg/kg bw/day was judged to be toxicologically not relevant because of the short duration immediately after the administration. The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of the last two weeks of gestation and during the lactation period. This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams. The mean daily food consumption was not adversely affected in male or female animals at 50, 150 and 300 mg/kg bw/day during the entire treatment period.


The examined parameters of the estrous cycle were comparable in all groups (control, 50, 150 and 300 mg/kg bw/day). The mean number of total birth, live born and alive pups was reduced at 300 mg/kg bw/day when compared to the actual control and historical control. A test item influence on the examined parameters of reproductive performance was not found at any dose level.


The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels. Macroscopic alterations related to the effect of the test item were not detected at 50, 150 or 300 mg/kg bw/day (male or female) at the necropsy examinations. The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 150 or 300 mg/kg bw/day. Quantitative examinations of ovaries revealed enhanced follicular atresia at 300 mg/kg bw/day with respect to their control, but no effects in primary, secondary or tertiary follicle counts.


The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 and 21. No adverse effects on extrauterine mortality, clinical signs, anogenital distance (male and female) or nipple retention (male) were detected. F1 generation (post-natal days 22-35) The body weight development and food consumption of the F1 animals was reduced at 150 and 300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. There were no clinical signs or necropsy findings in F1 generation (male and female) after the 14 days administration of 50, 150 or 300 mg/kg bw/day.


Based on these observations the NOAELs were determined as follows:


NOAEL for systemic toxicity of male/ female rats: 300 mg/kg bw/day


NOAEL for overall reproductive performance of male/ female rats: 150 mg/kg bw/day


NOAEL for F1 Offspring: 50 mg/kg bw/day

Endpoint:
extended one-generation reproductive toxicity - with developmental immunotoxicity (Cohorts 1A, 1B without extension, and 3)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
This information will be submitted later based on ECHA decision number CCH-D-2114493103-54-01/F.
Based on the results from the modified OECD 421 studies for the three peroxyesters only one study according OECD TG 443 with TBPND was initiated for the peroxyester group. This substance was chosen since ECHA requested for TBPND to include Cohort 3 (immuntoxicity, CCH-D-2114493102-56-01/F). The study results from this OECD TG 443 study will be used in a read-across grouping approach for the peroxyesters TBPIN and TBPPI to fulfil ECHAs requests on EOGRTS with these peroxyesters (CCH-D-2114493103-54-01/F and CCH-D-2114493105-50-01/F, respectively). This approach was also chosen because of animal welfare reasons.
The study according OECD TG 443 using the read-across substance TBPND (CAS 26748-41-4) is ongoing. The study report could not be completed by the deadline given by ECHA for this dossier update. As outlined in the attached document the final study report will be available end of April, 2022. Since additional time is needed for updating the technical dataset as well as the chemical safety report the submission of the requested dossier will be not later than June, 2022. Pre-liminary results are reported in the EPS for the source substance. As soon as the final report is available the dossier will be updated and a Read-Across Justification according RAAF (ECHA) for the grouping will be included.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Study not yet finalized. Study results will be reported in an update.
Critical effects observed:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Study not yet finalized. Study results will be reported in an update.
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Endpoint:
extended one-generation reproductive toxicity - with developmental immunotoxicity (Cohorts 1A, 1B without extension, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
15 July 2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks, as requested by ECHA.
- Basis for dose level selection: Based on a pre-test conduced with TBPND; furthermore, results from a subchronic toxicity study in rats and a OECD 422 study were taken into consideration for dose level selection.
- Exclusion of extension of Cohort 1B; not requested by ECHA based on available data
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B; not request by ECHA based on available data
- Inclusion of developmental immunotoxicity Cohort 3, as requested by ECHA
- Route of administration: oral (gavage)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P0 males and females: not older than 9 weeks
- Weight at study initiation: (P) Males: The weight variation in animals involved at the starting point of the study will not exceed ± 20 % of the mean group weight of each sex.
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
F1 offspring (after weaning): 2 animals of the same sex/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice
- Water: ad libitum, tap water
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30-70 %
- Air changes: > 10 per hr
- Photoperiod: 12/12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
Sunflower oil (Helianthi annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 25, 75 and 225 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and measured on 5 occasions. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
VEHICLE
- Justification for use and choice of vehicle: The test item is not stable in water. Therefore, sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 25, 75 and 225 mg/mL
- Amount of vehicle: 2 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sufficient stability and homogeneity in the chosen vehicle have been verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 ± 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL).
Duration of treatment / exposure:
Parental males were dosed for 70 days pre-mating and up to 14 days mating and until to the weaning of offspring. Overall treatment period is therefore up to 18 weeks for male parental animals.
Parental females were dosed for 70 days pre-mating, through up to 14 days mating period and throughout pregnancy and at least up to and including post-partum day 21 or up to the day before sacrifice. The day of birth (viz. when parturition is complete) is defined as day 0 post-partum. Overall treatment period is therefore up to 18 weeks for female parental animals.
Dosing of F1 offspring selected for follow-up examinations began on PND 22 up to post-natal day 90 (Cohort 1A) or at least up to post-natal day 97 (Cohort 1B).
F1 animals selected for determination of primary IgM antibody response to a T-cell dependent antigen in Cohort 3, were administered and observed individually up to and including the day before euthanasia on PND 61 ± 3.
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
No. of animals per sex per dose:
P0: 26
F1, Cohort 1A: 20
F1, Cohort 1B: 20
F1, Cohort 3: 20
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained in previous studies with TBPND in rats (OECD 422, OECD 421, OECD 408)
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter. Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21. Body weight of the female animals were additionally weighed on gestation day 10 in order to give accurate treatment volumes, but these data were not be evaluated statistically. F1 animals selected for follow-up examinations were weighed on post-natal day 22, then twice a week during the two weeks following weaning (on PND 25, 29, 32, 36, 39 and 42), and once weekly thereafter (Cohorts 1A, 1B and Cohort 3). For selected F1offspring, the body weight was recorded on the day when they attain puberty (completion of balano-preputial separation or vaginal patency). Fasted body weight was measured on the day of necropsy for all animals (P and F1)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption was determined weekly by reweighing the non consumed diet with a precision of 1 g during the treatment period except mating phase as follows:
- by weekly interval during premating and post-mating periods for P male animals and for F1 animals (Cohorts 1A, 1B and Cohort 3) after weaning;
- by weekly interval during premating period, on gestation days 0, 7, 14 and 21, on lactation days 0, 7, 14 and 21, then weekly if needed, for P female animals

WATER CONSUMPTION AND COMPOUND INTAKE: No

URINALYSIS: Yes
The following parameters were evaluated in selected test animals of P and F1 Cohort 1A generation:
Nitrite (NIT), pH, Glucose (GLUC), Urobilinogen (UBG), Bilirubin (BIL), Ketone (KET), Blood, Leucocytes (LEU), Specific Gravity (SG), Protein (PROT), Volume (VOL), Sediment (SED), Colour, Clarity

HEMATOLOGY/BLOOD COAGULATION
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
White blood cell (leukocyte) count (WBC), Red blood cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) hemoglobin (MCH), Mean Corpuscular (erythrocyte) hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count, Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

CLINICAL CHEMISTRY
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Sodium concentration (Na+), Potassium concentration (K+), Albumin concentration (ALB), Total protein concentration (TPROT)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- from 10 parent animals/sex/ group
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears from each parental female animal daily for two weeks before the mating starts.Vaginal smears were also prepared and estrous cycle was monitored daily during the mating period until evidence of copulation. Vaginal smear was prepared on the day of the necropsy of parental animals.Vaginal smears were examined for all F1 Cohort 1A females selected for follow-up examinations after the onset of vaginal patency until the first cornified smear is recorded thus determining the time interval between these events. Estrous cycle of F1 adult female animals was examined for a period of two weeks commencing on PND77 and PND84 in Cohort 1A and Cohort 1B, respectively, including necropsy days. Vaginal smears were stained with 1 % aqueous methylene blue solution. After drying, the smears were examined with a light microscope.
Sperm parameters (parental animals):
Sperm parameters were measured in all control and high dose male animals in P generation and in F1 generation in Cohort 1A. The one-side testes and epididymides were used for examinations. The weights of one-side testes and epididymides were determined and recorded. Sperm from the ductus deferens was collected for evaluation of sperm motility and morphology at the necropsy. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of the sperm motility, the mean percentage of motile sperms were determined. A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails). The epididymis was used for enumeration of cauda epididymis sperm reserves. The total number of sperm in homogenization was enumerated. The testis and epididymidis were frozen and enumeration were performed later.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups on PND13, FT3, FT4 and TSH in surplus pups at PND 4 (pooled by litters) and in pups not selected for Cohorts on post-natal day 22.

Sexual maturity of selected F1 animals was examined by observing balano-preputial separation (between post-natal days 25 and 35; or until PND40) or vaginal patency (between post-natal days 28 and 40; or until PND45). The body weight was determined on the day when balanopreputial separation or vaginal patency is completed

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

ASSESSMENT OF IMMUNOTOXICITY : Yes, Cohort 3

Animals of Cohort 3 were administered daily from weaning up to the day before the termination. The following observations will be conducted:
- Checking of mortality/ morbidity – twice daily;
- General clinical observations – daily;
- Detailed clinical observations were made outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
- Body weight measurement – on PND 22, then twice a week during the two weeks following weaning and once weekly thereafter;
- Food consumption – on PND 22 and weekly thereafter
- Blood sampling for T-cell dependent antibody response assay (TDAR) on PND 56±3 and PND 61±3;
On PND 61±3 the primary IgM antibody response to a T-cell dependent antigen – keyhole limpet hemocyanin (KLH) – is assessed after subcutaneous immunization of all Cohort 3 animals with KLH (0.2 mL at a concentration of 1.5 mg/mL on PND56±3; prior dosing with test-item). Anti-KLH IgM antigen concentrations in serum was measured before immunization and 5 days after immunization using a validated ELISA method (Rat Anti-KLH IgM ELISA kit, Life Diagnostics, Inc.). Responses typically peak five days after immunization.

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- in surplus pups at PND 4 (pooled by litters)
- from 10 adult F1 male and female animals/group (Cohort 1A ) at termination
- from 10 F1 pups/group not selected for cohorts on PND22
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: after mating period
- Maternal animals: on PND 22.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively, for high dose and control animals:

Organ weights (all parental (P) animal and all adult F1 animals of Cohort 1A):
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain
- liver
- kidneys
- heart
- spleen
- thymus
- pituitary
- thyroid glands (post-fixation)
- adrenal glands

The organs were fixed in 4% buffered formaldehyde solution. Paired organs were weighed together except for organs with macroscopically visible difference in size between the two organs. Absolute organ weight was recorded and reported.

Full histological examinations was performed on the above listed organs and tissues of control and high dose treated parental animals. Reproductive organs were examined in all animals suspected of reduced fertility (not mated, non pregnant or not delivered) in the low and mid dose group.

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS
At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) as follows:
- weighing of the lymph nodes associated with and distant from the route of exposure (submandibular and popliteal lymph nodes);
- splenic lymphocyte subpopulation analysis: CD4+ (Helper T cells) and CD8+ (Cytotoxic T cells) T lymphocytes, B lymphocytes and natural killer (NK) cells were identified by a validated flow cytometry method. Immuno-staining and immunophenotyping of spleen lymphocytes were carried out after preparation of single cell suspensions from one half of each provided spleen. For further information please refer to the attached background material.
Postmortem examinations (offspring):
SACRIFICE
- Offspring selected for Cohort 1A and Cohort 1B: on post-natal days 91and 98, respectively
- Offspring selected for Cohort 3 on PND 61±3
- Offspring not selected: on post-natal day 22

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weights ( all adult F1 animals of Cohort 1A):
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain
- liver
- kidneys
- heart
- spleen
- thymus
- pituitary
- thyroid glands (post-fixation)
- adrenal glands

In animals of Cohort 1B, the weight of following organs was determined:
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain,
- pituitary

For 10 male and 10 female pups per group, not selected for Cohorts, – from as many litters as possible – brain, spleen and thymus was weighed.

In animals of Cohort 3, the body weight and weight of following organs was determined:
- thymus
- spleen
- brain

Full histological examinations was performed on the above listed organs and tissues of control and high dose treated adult F1 animals in Cohort 1A. In adult F1 animals in Cohort 1B, the uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate (dorsolateral and ventral parts combined), seminal vesicles with coagulating glands as one unit (with their fluids), brain, pituitary will be processed to the block stage. In the ovaries of F1 adult females of control and high dose groups, a quantitative evaluation of primordial and small growing follicles, as well as corpora lutea was performed.

Detailed histological examination of testis was conducted with special emphasis on stages of spermatogenesis in the F1 male gonads and histopathology of interstitial testicular cell structure. Rete testis – where feasible – caput, corpus, and cauda of the epididymides were examined. All gross lesions were examined histologically. The fixed tissues were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Statistics:
The homogeneity of variance between groups was checked by Bartlett’s
homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of intergroup differences. Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Reproductive indices:
Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100

Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant):
Males: Number of males impregnating a females / Total number of males with confirmed mating x 100
Females: Number of pregnant females / Number of sperm positive females x 100

Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100
Offspring viability indices:
Formulas for Calculation of Pup Mortality and Sex Ratio Indices:

Post-implantation mortality: Number of implantations – Number of liveborns / Number of implantation x 100

Post-natal mortality: Number of liveborns – Number of live pups on PND 13 / Number of liveborns x 100

Survival Index: Number of live pups on PND 13 / Number of liveborns x 100

Sex ratio: Number of pups examined – Number of pups males (females) / Number of pups examined x 100
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Study not yet finalized. Study results will be reported in an update.
Key result
Critical effects observed:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Study not yet finalized. Study results will be reported in an update.
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
The study on ongoing and final results will be presented in an update. Preliminary findings are as follows:
Body weight: The body weight was lowered in male animals at 450 mg/kg bw/day (approximately -12 % terminally) but not in females.
Mating: The mating was successful: 26/26, 26/26, 26/26, 26/26 respectively to groups control, 50, 150, 450 mg/kg bw/day.
Pregnancy: Pregnancy was achieved in all groups with no difference compared to the control group: 25/26, 26/26, 24/26, 24/26, respectively to groups control, 50, 150, 450 mg/kg bw/day
Necropsy: 23/26 renal alteration (pale, enlarged or soft kidneys) in male animals at 450 mg/kg bw/day – thus, histological processing was performed on the kidneys of all animals.
Histopathology: chronic progressive nephropathy in male animals: 13/26 at 150 mg/kg bw/day and 26/26 at 450 mg/kg bw/day;
Based on daily observations, development of offspring was not essentially affected at any dose level. Evaluation, processing of additional data and histological processing of F1 animals are is in progress.
Based on first results for histopathology, the ovaries had a normal structure i.e., characteristic of the species, age and phase of the active sexual cycle in the control and 450 mg/kg bw/day groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well. The number of developing follicles and the number of follicular atresia was similar in the control and treated animals.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is considered acceptable without restrictions as it was conducted according to GLP regulations and OECD/EU guideline.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 421 (2010)


A Reproduction/Developmental toxicity screening test with the test item tert-Butylperoxy-3,5,5-trimethylhexanoat was performed according to OECD 421. The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to rats at repeated doses of 50, 160 and 500/400 mg/kg bw/day compared to control animals. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonad function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with administration of repeated doses. In this study, 12 animals/sex/dose were involved with exception of the 500/400 mg/kg bw group (13 males) and the 160 mg/kg bw/day group (13 females). All animals of the P generation were treated prior to mating (14 days) and throughout mating (16 days). Male animals were treated 17 days post mating. For females, treatment was continued through the gestation period and up to lactation day 3 or shortly thereafter, i.e. up to the day before the necropsy. Observations included mortality, clinical symptoms, body weight, food consumption, estrous cycle, mating, and pregnancy and delivery process.


Tert-Butylperoxy-3,5,5-trimethylhexanoat caused death of pregnant Hsd.Brl.HAN: Wistar rats at 160 and 400 mg/kg bw/day at the end of pregnancy and at early lactation due to acute-subacute tubular damage in the kidneys and related pulmonary alterations.


Reproductive performance of males was unaffected by treatment. Reproductive performance of female animals was affected by the treatment at 160 and 400 mg/kg bw/day through the parturition and nourishing and limited to the systemic toxicity at those doses. In the F1 generation, the higher mortality, clinical signs and depressed body weight development was considered to be the consequence of maternal toxicity at 160 and 400 mg/kg bw/day. Effects on reproduction and/or developmental of pups were thus only at doses pronounced in concentration with maternal toxicity and are in conclusion not regarded as relevant for indication for a specific hazard.


The NOAEL for tert-Butylperoxy-3,5,5-trimethylhexanoat for parental effects was 50 mg/kg bw /day. For reproduction parameters, the NOAELs were 400 mg/kg bw/day for male rats and 50 mg/kg bw/day for female rats. For F1 offspring, the NOAEL was 50 mg/kg bw/day.


Modified OECD 421 (2021)


A modified study according OECD TG 421 in rats was performed with TBPIN, in which the parent (P) generation was dosed 10 weeks prior to mating. For the peroxyesters TBPND (CAS 26748-41-4) and TBPPI (CAS 927-07-1) the same modified design was used in two additional OECD TG 421 studies. The rational was to compare the three peroxyesters regarding effects on reproductive performance after 10 weeks pre-mating exposure and to conclude on the possibility to perform only one subsequent OECD TG 443 study for all three peroxyesters (grouping approach). This 10-weeks pre-mating exposure is recommended in the OECD TG 443 study design. The results of all three modified OECD TG 421 studies are presented below:


TBPIN


Males received the test item or vehicle after mating up to the day before the necropsy (altogether for 98 days). Dams were additionally exposed through the gestation period and up to lactation days 21-22 (altogether for 114-122 days). Furthermore, one or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered from postnatal day 22 up to and including post-natal day 36.


Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 100 and 160 mg/kg bw/day doses corresponding to concentrations of 0, 10, 20 and 32 mg/mL. The application volume was 5 mL/kg bw. The doses were selected based on the OECD TG 421 study presented above and the OECD TG 408 study. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of test substance in the dosing formulations varied between the range of 99 % and 107 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 or 22 post-partum then were subjected to necropsy on post-partum day 22 or 23. Litters were weighed and offspring were observed for possible abnormalities. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 37. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-8 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 2-6 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Not mated, non-pregnant female animals and dams for which no living pups remained were sampled for thyroid hormone determination at termination, on Day 96 or 98.


All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one male animal at 100 mg/kg bw/day as well as the liver and kidneys of all animals were also preserved for a possible histological examination. Dam euthanized early in moribund state was subjected to necropsy on post-partum day 3 and all organs and tissues were preserved. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.


There was no test item related mortality at any dose level (50, 100 or 160 mg/kg bw/day by). One dam at 100 mg/kg bw/day was euthanized in moribund state on lactation day 3. Based on clinical observations and necropsy findings, large amount of blood loss at the parturition caused the poor condition of this dam. There were no similar findings at the higher dose, therefore the moribund state of this dam was considered to be a consequence of individual (not treatment related) disorder. Clinical observation Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (50, 100 or 160 mg/kg bw/day by) during the entire treatment period.


The body weight development was similar in male and female animals in the control and at 50, 100 and 160 mg/kg bw/day during the entire treatment period (pre-mating, mating and post-mating period for male animals; pre-mating, mating, gestation and lactation periods for female animals). Slight and transient changes in body weight gain at 160 mg/kg bw/day did not result in significant changes in the mean body weight in male or female animals during the pre-mating period (weeks 1 and 2).


The mean daily food consumption was not adversely affected in male or female animals at 50, 100 and 160 mg/kg bw/day during the entire treatment period. Estrous cycle The examined parameters of the estrous cycle were comparable in all groups (control, 50, 100 and 160 mg/kg bw/day).


There were no significant or dose related differences between the control and test item treated female animals in the examined parameters of the delivery (50, 100 and 160 mg/kg bw/day).


A test item influence on the examined parameters of reproductive performance was not found at any dose level. The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.


Necropsy examinations revealed macroscopic alterations presumably related to the effect of the test item in the liver (pale or yellowish-brown color, nut-meg-like pattern) in male animals at 100 or 160 mg/kg bw/day and in the kidneys (paleness and enlargement) in male animals at 160 mg/kg bw/day. Organ weight The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 100 or 160 mg/kg bw/day.


Quantitative examinations of ovaries did not reveal toxicologically relevant differences between the control and 160 mg/kg bw/day group.


The offspring’s development was not adversely influenced at any dose level. No adverse effects on mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.


Under the conditions of the present study, TBPIN administered at 50, 100 or 160 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats as far as investigated in this study. There were no signs of systemic toxicity in male or female animals at 50, 100 or 160 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 21 as far as investigated in this study after repeated oral administration of dams at 50, 100 or 160 mg/kg bw/day. There were no signs of systemic toxicity in male or female F1 animals at 50, 100 or 160 mg/kg bw/day after 14-day post-weaning treatment. Based on these observations the NOAEL were determined as follows:


NOAEL for systemic toxicity of male/ female rats: 160 mg/kg bw/day


NOAEL for reproductive performance of male/ female rats: 160 mg/kg bw/day


NOAEL for F1 Offspring: 160 mg/kg bw/day


TBPND


A modified OECD 421 study in rats was performed. The purpose of this study was to determine the dose levels for the main study (OECD 443) and obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 50, 150 and 300 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 150 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 150 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPND in the dosing formulations varied between the range of 95 % and 109 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 99 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114-127 days). Non-pregnant female animals were administered for 96 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to days 21 post-partum then were subjected to necropsy on post-partum days 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-8 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals were sampled for thyroid hormone determination at termination, on Day 96.


All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, thyroid glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one female animal at 50 mg/kg bw/day, liver, spleen, heart, lungs in one dam at 150 mg/kg bw/day as well as the kidneys of some animal were also preserved for a possible histological examination. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. A quantitative examination of ovaries was performed in all female animals in the control and high dose groups.


There was no test item related mortality at any dose level (50, 150 or 300 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (50, 150 or 300 mg/kg bw/day) during the entire treatment period. Salivation observed in male animals at 300 mg/kg bw/day was judged to be toxicologically not relevant because of the short duration immediately after the administration. The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of the last two weeks of gestation and during the lactation period. This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams. The mean daily food consumption was not adversely affected in male or female animals at 50, 150 and 300 mg/kg bw/day during the entire treatment period.


The examined parameters of the estrous cycle were comparable in all groups (control, 50, 150 and 300 mg/kg bw/day). The mean number of total birth, live born and alive pups was reduced at 300 mg/kg bw/day when compared to the actual control and historical control. A test item influence on the examined parameters of reproductive performance was not found at any dose level.


The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels. Macroscopic alterations related to the effect of the test item were not detected at 50, 150 or 300 mg/kg bw/day (male or female) at the necropsy examinations. The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 150 or 300 mg/kg bw/day. Quantitative examinations of ovaries revealed enhanced follicular atresia at 300 mg/kg bw/day with respect to their control, but no effects in primary, secondary or tertiary follicle counts.


The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 and 21. No adverse effects on extrauterine mortality, clinical signs, anogenital distance (male and female) or nipple retention (male) were detected. F1 generation (post-natal days 22-35) The body weight development and food consumption of the F1 animals was reduced at 150 and 300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. There were no clinical signs or necropsy findings in F1 generation (male and female) after the 14 days administration of 50, 150 or 300 mg/kg bw/day.


Based on these observations the NOAELs were determined as follows:


NOAEL for systemic toxicity of male/ female rats: 300 mg/kg bw/day


NOAEL for reproductive performance of male/ female rats:150 mg/kg bw/day


NOAEL for F1 Offspring: 50 mg/kg bw/day


TBPPI


A modified OECD 421 study in rats was performed with the grouping substance TBPPI (CAS 927-07-1). The purpose of this study was to obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 250 and 350 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 250 and 350 mg/kg bw/day doses corresponding to concentrations of 0, 50 mg/mL, 125 mg/mL and 175 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPPI-75-AL in the dosing formulations varied between the range of 90 % and 108 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 100 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114, 115, 118 or 125 days). Non-pregnant female animals and dam not delivered were administered for 95, 97, 98 or 102 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 - 23 post-partum then were subjected to necropsy on post-partum day 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) from 2-5 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-6 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals and dam not giving birth were sampled for thyroid hormone determination at termination on the day of the necropsy (Days 95, 97, 98 or 102). All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands and thyroid glands of all adult animals were preserved. In addition, organs with macroscopic findings were also preserved for a possible histological examination. A quantitative examination of the ovaries was performed in all female animals in the control and high dose groups. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.


There was no mortality at any dose level (100, 250 or 350 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (100, 250 or 350 mg/kg bw/day) during the entire treatment period. Salivation and nuzzling up the bedding material observed at 250 and 350 mg/kg bw/day were with short duration after the administration of the test item therefore these signs were considered to be toxicologically not relevant.


The body weight development was depressed in male animals at 250 and 350 mg/kg bw/day during the pre-mating, mating and post-mating periods. The difference with respect to the control was lower than 10 % at 250 mg/kg bw/day therefore was judged to be toxicologically not relevant.


The mean daily food consumption was slightly reduced in male animals at 350 mg/kg bw/day in accordance with the body weight changes during the entire treatment period. Estrous cycle The examined parameters of the estrous cycle were not adversely affected in any group (control, 100, 250 and 350 mg/kg bw/day). Delivery data of dams There were no significant or dose related differences between the control and test item treated female animals in the examined parameters of the delivery (100, 250 and 350 mg/kg bw/day).


A test item influence on the examined parameters of reproductive performance was not found at any dose level. Serum thyroid hormones The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.


Necropsy examinations did not reveal specific macroscopic alterations related to the effect of the test item in male or female animals at 100, 250 or 350 mg/kg bw/day.


The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 100, 250 or 350 mg/kg bw/day.


There were no toxicologically relevant differences in the mean number of primordial, primary, secondary, tertiary follicles, atresia or corpora lutea at 350 mg/kg bw/day at the quantitative examination of ovaries.


The offspring’s development was not adversely influenced at any dose level. No adverse effects on mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected. There were no clinical signs, changes in body weight or mean daily food consumption or necropsy findings in F1 generation (male and female) after the 14 days administration of 100, 250 or 350 mg/kg bw/day.


Based on these observations the NOAELs were determined as follows:


NOAEL for systemic toxicity of male rats: 250 mg/kg bw/day


NOAEL for systemic toxicity of female rats: 350 mg/kg bw/day


NOAEL for reproductive performance of male/ female rats: 350 mg/kg bw/day


NOAEL for F1 Offspring (PN 0-21): 350 mg/kg bw/day


NOAEL for F1 generation (PN 22-35): 350 mg/kg bw/day


Overall Conclusion


In the OECD TG 421 study performed in 2010, higher mortality, clinical signs and depressed body weight development for F1 offspring was considered to be the consequence of maternal toxicity, which was observed in this study at 160 and 400 mg/kg bw/day.


10-week pre-mating exposure to TPBIN in a modified OECD TG 421 study does not adversely affect reproductive performance and fertility of male and females rats up to the highest dose of 160 mg/kg bw/day. For the peroxyesters TBPPI and TBPND the results for the modified OECD TG 421 studies also show no adverse effects on the parameters of reproductive performance of male and female rats. This 10-weeks pre-mating exposure is recommended in the OECD TG 443 study design. For all three peroxyesters, no adverse effects on estrous cycle and ovaries (follicle counts) have been observed in the modified OECD 421 studies. The development of the F1 was not adversely effected after exposure to TPBIN and TBPPI in the modified OECD TG 421 studies up to the highest doses tested. For TBPND the body weight development of the offspring was depressed up to PND 36 at mid and high dose.


Based on the results from the modified OECD 421 studies for the three peroxyesters only one study according OECD TG 443 with TBPND was initiated for the peroxyester group. This substance was chosen since some effects on F1 offspring have been reported for TPBND, which was not the case for the two other peroxyesters. Therefore, read-across to the initiated OECD TG 443 study performed with TBPND will be a worst case approach. Furthermore,  ECHA requested for TBPND to include Cohort 3 (immuntoxicity, CCH-D-2114493102-56-01/F). The study results from this OECD TG 443 study will be used in a read-across grouping approach for the peroxyesters TBPIN and TBPPI to fulfil ECHAs requests on EOGRT studies with these peroxyesters (CCH-D-2114493103-54-01/F and CCH-D-2114493105-50-01/F, respectively). This approach was also chosen because of animal welfare reasons. The OECD TG 443 study with TBPND will be completed by June 2022 and a dossier update will be submitted as soon as the study results are available.

Effects on developmental toxicity

Description of key information

Under the conditions of a guideline and GLP compliant study, the no observed effect level (NOEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat for maternal toxicity was 150 mg/kg bw /day and for developmental toxicity was 20 mg/kg bw/day for male and female rats. The no observed adverse effect level (NOAEL) for maternal and developmental toxicity was 150 mg/kg bw/day for male and female rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is considered acceptable without restrictions as it was conducted according to GLP regulations and OECD/EU guideline.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 rat


Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with TBPIN by oral administration daily at three dose levels of 20, 50 and 150 mg/kg bw/day from day 6 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 ml/kg/bw. During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 19, 20, 21 and 22 evaluated litters in the control, 20, 50 and 150 mg/kg groups, respectively. Two pregnant females died in the course of the study, one female in the 50 mg/kg bw/day group on gestational day 17 and one in the 150 mg/kg bw/day dose group on gestational day 9. The death and clinical signs such as reduced body tone and reduced activity shortly before death of the female in the mid dose group and the death and necropsy findings (brownish-yellowish discolouration in the whole lung tissue and dark areas in the liver) of the dam at 150 mg/kg bw/day were considered to be likely without a relationship with an effect of the test item. There were no clinical signs and necropsy findings recorded for the survived dams in the experimental groups. There was no indication of an effect of the test item on body weight, corrected body weight and food consumption of the dams in the 20, 50, 150 mg/kg bw/day dose groups. There was no effect indicated related to the administration of TBPIN in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The mean fetal weight, was similar in the control and 20 mg/kg bw/day groups. The slight but statistically significant reduction in the bodyweight of the fetuses in the 50 mg/kg bw/day and 150 mg/kg bw/day dose groups might be attributed to an effect of the test item. Since fetal weights were still within the range of the laboratory´s historical control data the weight reduction was considered as non-adverse. Relative placental weight was similar in all experimental groups.


The increased incidence of external variations i.e. growth retarded fetuses in the 150 mg/kg bw/day dose group was within the laboratory´s historical control range and thus, considered as non-adverse.


The presence of the fetal malformations found at external examination (one in the control and one in the high dose group) was considered to be incidental.


The visceral malformations were considered to be incidental and were not attributed to the administration of the test item to the dams. The incidence of visceral variations was similar to the vehicle control level.


Skeletal malformations in two fetuses of the low dose group and in one fetus of the control group were without a relationship to the test item. Statistically significant skeletal variations were seen in the 20 and in the 150 mg/kg bw/day dose group when the ossification of sternum (3 or less ossified sternebra) was evaluated. Considering that there was no dose response indicated between the low and mid dose group and that the mean percent of the high dose group was slightly above the laboratory´s historical control level, this variation was judged to be in the biological variations.


Incomplete ossification of the skull-bones (marked and less marked) was evaluated as variation during skeletal examination. Incomplete ossification of the skull bones correlated with the slightly lower mean fetal weight observed at 50 and 150 mg/kg bw/day dose groups, which was below the mean value seen in the laboratory´s historical control data. However, the data pool of laboratory´s historical control data comprises only a few studies and thus, it is not considered that the whole range of this rat strain is reflected. In general, retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al., 2005). The maximum incidence of incomplete ossification of 8% observed in this study is within the normal range reported for rats (e.g. Mylchreest et al., 2005, RMS France, 2007). Moreover, since the incomplete ossification was observed in skull bones and not in bones that are normally well ossified the effect is classified as a low significant finding which may not be adverse according to Carney and Kimmel (2007). Overall, incomplete ossification of the skull bones (marked and less marked) seen at 50 mg/kg bw/day and at 150 mg/kg bw/day is considered to be non adverse.


 


Based on these observations the No Observed Effect Level (NOEL) was determined as follows:


NOELmaternal toxicity: 150 mg/kg bw/day


NOELdevelopmental toxicity: 20 mg/kg bw/day


 


Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:


NOAELmaternal toxicity: 150 mg/kg bw/day


NOAELdevelopmental toxicity: 150 mg/kg bw/day


 


REFERENCES


- Carney, E.W. and C.A. Kimmel (2007): Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res., 80:473-496


- Mylchreest, E.et al. (2005): Evaluation of the developmental toxicity of 8-2 Telomer B Alcohol.Drug and Chemical Toxicology., 28:315-328


- Rapportour Member State (RMS) France (2007): Draft Assessment Report, Initial risk assessment provided by the rapporteur Member State France for the existing active substance Fluazifop-P-Butyl of the third stage (part A) of the review programme referred to the Article 8(2) of Council Directive 91/414/EEC.Volume 3, Annex B, part 4, B.8

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is not classified and labelled reproduction/developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.

Additional information