Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In accordance with column 2 of REACH Annex VIII, the performance of a reproduction/developmental toxicity screening test (OECD guideline 421) is not required since a prenatal developmental toxicity study is available for assessment, and sufficient data are available (histopathology data and organ weights from a 90 day repeated dose toxicity study) to assess the toxicity to reproduction.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

REACH allows the assessment of the reproductive toxicity of a given chemical with the help of findings from studies with repeated administration. This is in line with the idea that the information requirements under REACH are regarded as the evaluation of endpoints which does not necessarily require data from specific studies. Because of a high correlation, histopathology data and organ weights from repeated dose studies may be used to assess male fertility (Mangelsdorf, 2003). These parameters, taken from 90 day studies, were in fact shown to be more sensitive than fertility parameters that were measured during multi-generation studies. It could also be shown that exposure for 4 weeks suffices for an assessment of male fertility, although 90 day studies have been regarded as superior in the past because they cover a complete cycle of spermatogenesis (Mangelsdorf, 2003). If such a 28 day study shows neither relevantly elevated testis or ovary weights nor histopathological alterations in those organs, the weight of the evidence is that effects on reproduction are also not expected (BAuA Forschungsbericht Fb 984, 2003). A comparison of more than one hundred 90 day studies with two generation studies that used the same test substance additionally showed that the NOAELs differed by less than the variation limit of studies, i.e. a factor of two (Janer, 2007). Therefore, the information gained from a reproduction/developmental screening test concerning toxicity to reproduction can be regarded as minimal if a 90 day study has been performed.

In a subchronic oral study in rats, Triisobutyl phosphate (TIBP) was administered at target exposure levels of 0, 200, 1000 or 5000 parts per million (ppm) in feed for approximately 13 weeks. (Monsanto, 1990). Groups consisted of 30 rats/sex for the 0 and 5000 ppm dose levels and 10 rat/sex for the remaining levels. Treatment-related changes were limited to decreased food consumption at initiation of treatment, a decrease in neutrophil count and an increase in plasma cholesterol (males only) at 5000 ppm. The weight of the testes and epididymides, and post-mortem and histological examination of the following organs revealed no treatment-related effects: ovaries, prostate, seminal vesicles, skin (with mammary tissue), testes, epididymides, uterus (corpus and cervix).

Taken together, sufficient data are available to assess toxicity to reproduction of Triisobutyl phosphate (a reliable teratogenicity study that showed no treatment-related effects, and histopathology data and organ weights from a 90-day repeated dose toxicity study). Therefore, and for the sake of animal welfare, the performance of an additional reproduction/developmental toxicity test (OECD guideline 421) is not warranted.

Effects on developmental toxicity

Description of key information

In an oral developmental study performed with rats (according to OECD guideline 414), the NOAEL for maternal toxicity was 300 mg/kg bw/day based on based on retardation of bodyweight gain and increased water consumption. The NOAEL for developmental toxicity was 1000 mg/kg bw/day (highest dose tested).

In an oral prenatal developmental study performed with rabbits (according to OECD guideline 414), the NOAEL for maternal and developmental toxicity was 150 mg/kg bw/day due to the absence of adverse effects.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan - Apr 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 18200055
- Expiration date of the lot/batch: 30 Jun 2020
- Purity: 98.3 g/100 g
- Physical description: Clear colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light
- Stability at higher temperatures: Avoid temperatures > 70 °C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) will be homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations will be prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations will be kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle will be continuously stirred until and during dosing. Adjustment will be made for specific gravity of the vehicle and test item.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : dosing formulation (w/w) in arachis oil
Species:
rabbit
Strain:
New Zealand White
Remarks:
outbred, SPF-quality
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: approximately 17 - 19 weeks
- Weight at study initiation: approximately 3000 to 4300 g
- Housing: On arrival females were individually housed in labelled cages with perforated floors. The cages were equipped with water bottles.
- Diet (e.g. ad libitum): Free access to pelleted diet for rabbits, except during designated procedures. In addition, pressed hay was provided during the study period.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles / containers.
- Acclimation period: at least 2 days prior to the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
The following vehicles were tested and did not result in a homogeneous formulation:
- Elix water (does not mix)
- 1% CMC with 0.1% Tween80 (50% formulation; does not mix)
- 1% CMC with 0.1% Tween80 (20% formulation; does not mix)
- 0.5% CMC with 1.25% Tween80 (50% formulation; does not mix)
- 0.5% CMC with 1.25% Tween80 (20% formulation; does not mix)

The following vehicles were tested and did result in an acceptable formulation (solution):
- Propylene glycol (20% formulation)
- Arachis oil (20, 50, 80 and 90% formulations)
- Corn oil (20 and 50% formulations)

Propylene glycol cannot be used in rabbit developmental toxicity studies, as according to data of the test faclility propylene glycol causes adverse effects on the development of rabbits. When choosing between corn oil and arachis oil, arachis oil is preferred as it appears to be better tolerated by the rabbit strain used.
Based on the trial results, the test substance has been administered to the animals by oral gavage formulated in arachis oil.

- Concentration in vehicle: 0, 15, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 1 mL/kg dose volume
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
For the formulation of Group 2 prepared for use in Week 1, the mean accuracy was below the target concentration (i.e. 86% of target). An out of specification process was started but no analytical reason could be found. Therefore, a new occasion for Group 2 was performed in Week 4; results of these analysis were within the criterion range of 90 - 110 %.
A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 1. The maximum contribution to the Group 2 samples was 0.0020 %. It was considered that it had no significant effect on the analysed concentrations in the test samples.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
23 days (7 days a week from day 6 to day 28 post-coitum, inclusive)
Frequency of treatment:
once daily, 7 days a week
Duration of test:
subacute (26 days)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of the dose range finder, and in attempt to produce graded responses to the test item. In this study, dose levels of 100 and 300 mg/kg/day were administered:
• All pregnant animals at 300 mg/kg presented with up to moderate body weight loss (1 - 6 %) over days 6 - 9 post-coitum. Mean body weight gain was reduced when compared with control group over the entire treatment period, resulting in a 7 % lower mean body weight at the end of the study period (on day 29 post-coitum). Mean relative food consumption was up to severely reduced vs control over days 6 - 15 post-coitum (up to 91 %). From day 15 - 18 post-coitum onwards, mean food intake started to partially recover, followed by a recurrent decrease at the end of the treatment period.
• At 100 mg/kg, absent mean body weight gain was observed over days 6 - 9 post-coitum, which was mainly attributed to 3 out of 6 animals that presented with up to 3 % body weight loss over that period. A slight reduction in mean body weight (2.5 %) vs control was observed at the end of the treatment period (on day 29 post-coitum). Mean relative food consumption was reduced (up to 42 %) vs control from start of treatment until day 15 - 18 post-coitum, followed by a partial recovery afterwards.
Based on these results, and after consultation with the Sponsor, a maximum dose level of 150 mg/kg/day was selected for the current study.
The high-dose level should produce some maternal and / or developmental toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
Maternal examinations:
MORTALITY / MORIBUNDITY CHEKCS: Yes
- Time schedule: At least twice daily throughout the study.
- Procedure: Animals will be observed for general health / mortality and moribundity. Animals will not be removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From day 6 post-coitum onwards up to the day prior to necropsy, animals will be observed at least once daily.
- Animals will be observed for specific clinical signs. The time of onset, grade and duration of any observed signs will be recorded. Signs will be graded for severity and the maximum grade will be predefined at 3 or 4. Grades will be coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) will be scored. In the data tables, the scored grades will be reported, as well as the percentage of animals affected in summary tables. Cage debris will be examined to detect premature birth, if applicable.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals will be weighed on days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In order to monitor the health status animals will be weighed more often.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption of animals will be measured for days 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 - 24, 24 - 27 and 27 - 29 post-coitum.
- Procedure: Food consumption will be quantitatively measured.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Regular basis throughout the study.
- Procedure: Water consumption will be monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 post-coitum
- Organs examined: All animals will be subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities will be recorded, collected ad fixed in 10 % buffered formalin; collection of specific macroscopic abnormalities may be omitted only at the discretion of the Study Director. No tissues, except the uterus, will be weighed. Each ovary and uterine horn of all animals will be dissected and examined as quickly as possible to determine the following as part of the necropsy procedure:
• The number of corpora lutea.
• The weight of the uterus (not for animals found dead, sacrificed before planned necropsy or that delivered early).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: The number and distribution of live and dead fetuses. The number and distribution of embryo-fetal deaths.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No
Statistics:
All statistical tests will be conducted at the 5 % significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1 % or 5 % levels.
Numerical data collected on scheduled occasions will be analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation will be reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics will be performed according to the matrix below when possible, but will exclude semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons will be made:
• Group 2 vs. Group 1
• Group 3 vs. Group 1
• Group 4 vs. Group 1

PARAMETRIC
Datasets with at least 3 groups (the designated control group and 2 other groups) will be compared using Dunnett-test (many-to-one-t-test).

NON-PARAMETRIC
Datasets with at least 3 groups will be compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution will be compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation will be subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA reveals statistically significant (p < 0.05) intergroup variance, Dunn's test will be used to compare the coumpound-treated groups to the control group.

INCIDENCE
An overall Fisher's exact test will be used to compare all groups. The above pairwise comparisons will be conducted using Fisher's exact test whenever the overall test is significant.
Indices:
- Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites)/(number of corpora lutea)) * 100
- Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/(number of implantation sites)) * 100
- Viable fetuses affected / litter (%) = ((number of viable fetuses affected / litter)/(number of viable fetuses / litter)) * 100
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted during the observation period.
Transient red fluid on the manure tray (slight) was observed in one control (No. 04), one animal at 50 mg/kg/day (No. 53), and 2 animals at 150 mg/kg/day (Nos. 71 and 85) on a single day over the entire study period (Day 20 or 25 post-coitum). At the incidence observed (only in 1-2 animals per group on a single day, including the control group), this observation was considered to be unrelated to treatment. Additionally, one animal at 15 mg/kg/day (No. 38) presented with a red secretion from the vagina on Day 18 post-coitum. At the incidence observed (only in a single low dose animal), it was considered to be a chance finding.
Reduced faeces production (up to severe) was observed in animals among all groups, including controls, with no clear dose-related response.
Piloerection and pale appearance (slight) were observed in one animal at 150 mg/kg/day (No. 70) for 1-2 days at the end of the treatment period (Day 27-28 post-coitum). Similar observations were recorded in one control animal (No. 16) that was noted with piloerection for 8 consecutive days during the last week of treatment and lean appearance at the end of the treatment period (Day 28 post-coitum) together with transient mucus in the faeces for 2 consecutive days (Days 12-13 post-coitum). At the incidence observed, and as no doserelated
response was observed, these observations were considered not to be related to treatment with the test item.
Before the start of treatment, 2 rabbits were noticed without toe(s) in one paw: one animal at 150 mg/kg/day (No. 67) presented with absent fingers in the left front paw and one control animal (No. 18) was lacking one finger in the right front paw. Since this observation was noticed before start of treatment, this clinical sign was unrelated to treatment.
Incidental findings that were noted included scabs, wounds, flew swelling, alopecia and broken teeth. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions
in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
There were 2 preterm decedents over the study period:
One animal at 150 mg/kg/day (No. 81) was sacrificed in extremis on Day 23 post-coitum for animal welfare reasons as she presented with persistent body weight loss (up to 6% vs start of dosing) and up to severely reduced relative food consumption from start of treatment
onwards. She was noted with persistent moderate/severe reduction in faeces production for more than 2 consecutive weeks (since Day 9 post-coitum onwards) and she was additionally noted with piloerection on Day 23 post-coitum. At necropsy, no macroscopic findings were
noted and she presented with gravid uterus (7 alive fetuses). The single female at 150 mg/kg/day sacrificed in extremis occurred within the normal limits for rabbits of this strain and age housed in the same conditions as in this study. One animal at 15 mg/kg/day (No. 23) died after dosing on Day 28 post-coitum. Immediately after dosing on the day of death, she was noted with blood in the nose and moribund (blood was also observed in the catheter used for oral gavage). Normal body weight gain and food consumption were observed in this animal: only a transient and moderate decrease in relative food consumption over Days 12-15 post-coitum, with a complete recovery thereafter, and a transient reduction in faeces production (up to moderate) on several days were recorded for this animal. At necropsy, she presented with gravid uterus (7 alive fetuses and 2 late resorptions) and macroscopic findings indicative of an oral misgavage were recorded (i.e. perforation of the trachea and esophagus). Based on the observations recorded after dosing and at necropsy, and as any signs of severe toxicity were observed for this animal the previous days, this spontaneous death was regarded as related to the oral gavage procedure and, as such, unrelated to treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No relevant changes in mean body weight and body weight gain were observed after treatment up to 150 mg/kg/day.
At the beginning of the treatment period (over Days 6-9 or 6-12 post-coitum), some individual animals across all groups, including controls, presented with absent body weight gain or slight body weight loss (up to 2%), which was considered unrelated to treatment as no
dose response was observed.
Some individual animals at 50 mg/kg/day (Nos. 49) and 150 mg/kg/day (Nos. 70 and 77) were noted with a moderate body weight loss (6-7%) towards the end of the treatment period (over Days 21-24 to 29 post-coitum) with one animal at 50 mg/kg/day (No. 53) presenting with a 4% body weight loss on Day 29 post-coitum vs start of dosing. At the incidence observed and in the absence of a dose response, these findings were considered not toxicologically relevant.
In addition, body weight loss (slight/mild) at several intervals was observed for several animals across all groups, including controls, without a clear dose relationship.
The slightly lower (2%) in mean body weight (before and after correction for gravid uterus weight5) observed at 50 mg/kg/day at the end of the study period was considered a chance finding in the absence of a dose-related response.
Mean body weight gain corrected for the weight of the gravid uterus was unaffected by treatment up to 150 mg/kg/day. All mean values were within the range of the historical control data.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No relevant changes in mean food consumption (before or after allowance for body weight) were observed after treatment up to 150 mg/kg/day.
The slightly lower (non-significant) in food intake observed at 50 mg/kg/day at the end of the treatment period was considered incidental as no dose response was observed. In addition, at this mid dose level, 2 animals (Nos. 53 and 57) presented with severely reduced
food consumption (<10 g/kg body weight/day) for ≥ 4 consecutive periods, without showing severe body weight loss when compared with start of treatment.
In addition, one animal each at 15, 50 and 150 mg/kg/day (Nos. 30, 55 and 67) were noted with reduced relative food consumption (<20 g/kg body weight/day) over almost the entire treatment period (from Day 6 or 12 post-coitum onwards). In the absence of a dose-related
response, and as similar effects were recorded in several control animals (i.e. Nos. 6 and 8), these findings were considered to be incidental.
In general, a lower relative food consumption (up to severe) was observed at several intervals for several animals across all groups, including controls, without a clear relationship to dose.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea and implantation sites were within the range of the historical control data in all groups. At 50 mg/kg/day, the mean litter incidence of preimplantation loss was increased vs concurrent control (15.9% vs 5.2%) and was also slightly higher than the historical control 95th percentile (P95=15.3%, max value=17.2%). As no dose-related response was observed, it was considered to be a chance finding.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 5 females were not pregnant in this study: 2 females at 15 mg/kg/day (A037 and A039) and 50 mg/kg/day (A059 and A062), and 1 female at 150 mg/kg/day (A080). All pregnant females had litters with viable fetuses. The percentage of pregnant females was in the range of normal biological variation.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: absence of adverse effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 150 mg/kg/day.
Mean combined (male and female) fetal body weights were 38.4, 37.1, 39.1 and 38.7 gram for the control, 15, 50 and 150 mg/kg/day groups, respectively.
The mean female fetal weight at 15 mg/kg/day (35.8 g) was lower than the concurrent control mean (not statistically significant) and slightly below the 5th percentile of the historical control data (P5=36.1 g, min value=36 g). As no dose-response was observed, this finding was considered to be a chance finding.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 150 mg/kg.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size upon treatment up to 150 mg/kg/day.
There were 3 control litters (A011, A014 and A016) and 1 litter at 150 mg/kg (A068) that presented with 1 dead fetus each. The dead fetus in the high dose group was considered to be unrelated to treatment, at the incidence observed (well within the historical control range
P5-P95=0.0-1.0%/litter, max value=1.5%/litter) and as it was also noted in the concurrent control group.
Mean litter sizes were 8.6, 9.1, 8.2 and 9.5 fetuses/litter for the control, 15, 50 and 150 mg/kg/day groups, respectively. All mean values (absolute and % per litter) were within the range of the historical control data.
The mean litter incidence of post-implantation loss was within the range of available historical control data in all groups. The higher litter incidence of late resorptions observed at 15 mg/kg/day, slightly above the historical control 95th percentile (P95=4.2%), was attributed to an individual value (A032). In the absence of a dose-response relationship, this was regarded as unrelated to treatment.
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 150 mg/kg/day.
External malformations occurred in 2 (2), 0 (0), 1 (1) and 2 (2)7 fetuses (litters) in the control, 15, 50 and 150 mg/kg/day groups, respectively. At the high dose, fetus A085-08 had a bilateral carpal flexure without underlying skeletal anomaly and the dead fetus in litter A068 had an omphalocele. This latter fetus, A068-01, presented also with one visceral and skeletal malformation; abnormal lobation of the lung and fusion of the frontal bones.
The malformed fetus at 50 mg/kg/day, A056-06, had a cleft palate together with multiple viscerally abnormalities (namely hydrocephaly, small stomach, narrow aortic arch, ventricular septum defect, dilated pulmonary trunk, small lung lobes and an abnormal lung lobation). Skeletal examination of this fetus substantiated the palate cleft and revealed two skeletal abnormalities; fused skull bones and a sternal anomaly.
The two affected control fetuses either had anencephaly (A022-08) with underlying skeletal abnormalities or a distended abdomen (A018-03) with multiple visceral malformations namely ascites, a large heart and bilateral malpositioned testes.
Due to the single occurrence and/or occurrence in control group, these malformations were considered to be chance findings.
No external variations were observed in this study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 150 mg/kg/day.
Skeletal malformations occurred in 4 (4), 3 (3), 4 (4) and 1 (1) fetuses (litters) in the control, 15, 50 and 150 mg/kg/day groups, respectively.
Besides the skeletal malformations described above in fetus A056-06 and dead fetus A068-01 (of the 50 and 150 mg/kg/day groups, respectively) with an external malformation, two fetuses at 50 mg/kg/day (A049-09 and A064-09) had a rib anomaly and another fetus at this dose level (A061-01) had a vertebral anomaly. A vertebral anomaly with or without associated rib anomaly was also observed in three fetuses at 15 mg/kg/day (A024-03, A028-05 and A032-05) and in three control fetuses (A014-06, A015-05 and A022-01). One of these control fetuses (A015-05) also had a sternal anomaly and another control fetus (A018-07) had a vertebral centra anomaly. Because these malformations occurred singly, in concurrent controls, without a dose-related trend and/or were noted previously in historical controls, they were not considered to be treatment related.
Skeletal variations occurred at an incidence of 81.2%, 74.9%, 76.5% and 80.2% per litter in the control, 15, 50 and 150 mg/kg/day groups, respectively. All the variations noted were considered not to be treatment related as they occurred infrequently, in the absence of a doserelated
incidence trend, in control fetuses only and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 150 mg/kg/day.
Visceral malformations occurred in 1 (1), 0 (0), 2 (2) and 2 (2)8 fetuses (litters) in the control, 15, 50 and 150 mg/kg/day groups, respectively.
Besides the visceral malformations described above in fetus A018-03, A056-06 and dead fetus A068-01 (of the control, 50 and 150 mg/kg/day groups, respectively) with an external malformation, one fetus at 150 mg/kg/day (A084-02) was noted with tetralogy of Fallot and one fetus at 50 mg/kg/day (A057-07) had a malpositioned kidney. The single occurrence and group distribution of these malformations did not indicate a treatment relationship and, therefore, these findings were considered to be spontaneous in origin.
Visceral variations occurred at an incidence of 10.3%, 7.7%, 10.4% and 8.0% per litter in the control, 15, 50 and 150 mg/kg/day groups, respectively. All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 3: Summary of female body weights, F0 generation

 

 

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

POST COITUM

DAY 6

 

MEAN

 

3618

 

3561

 

3577

 

3582

 

ST.DEV.

350.5

305.2

315.0

336.0

 

N

22

20

20

21

DAY 9

MEAN

3664

3658

3645

3649

 

ST.DEV.

331.1

316.2

312.6

305.0

 

N

22

20

20

21

DAY 12

MEAN

3718

3722

3705

3717

 

ST.DEV.

317.1

308.3

315.7

310.9

 

N

22

20

20

21

DAY 15

MEAN

3761

3787

3749

3783

 

ST.DEV.

309.5

318.5

305.7

302.6

 

N

22

20

20

21

DAY 18

MEAN

3818

3844

3752

3823

 

ST.DEV.

329.8

314.0

320.7

300.9

 

N

22

20

20

21

DAY 21

MEAN

3822

3848

3775

3857

 

ST.DEV.

328.1

310.7

316.2

309.5

 

N

22

20

20

21

DAY 24

MEAN

3836

3879

3803

3917

 

ST.DEV.

306.4

304.6

317.8

241.9

 

N

22

20

20

20

DAY 27

MEAN

3864

3907

3799

3954

 

ST.DEV.

321.4

311.0

332.1

223.4

 

N

22

20

20

20

DAY 29

MEAN

3903

3953

3815

3983

 

ST.DEV.

309.0

325.5

329.8

216.8

 

N

22

19

20

20

Table 4: Summary of macroscopic findings in females of F0 generation

 

 

GROUP 1 CONTROL

 

GROUP 2

15 MG/KG

 

GROUP 3

50 MG/KG

 

GROUP 4

150 MG/KG

 

POST COITUM

Animals examined

 

22

 

22

 

22

 

22

Animals without findings

18

20

20

20

Animals affected

4

2

2

2

Trachea Perforation(s)

 

0

 

1

 

0

 

0

Esophagus

 

 

 

 

Perforation(s)

0

1

0

0

Stomach

 

 

 

 

Focus/foci

1

0

0

0

Liver

 

 

 

 

Nodule(s)

0

0

1

0

Spleen

 

 

 

 

Ectopic splenic tissue

1

0

0

0

Skin

 

 

 

 

Alopecia

1

0

1

1

Bone

 

 

 

 

Broken

0

1

0

0

Organ missing

1

0

0

0

Agenesis

0

0

0

1

 

Table 5: Summary of maternal survival and pregnancy status

Dose group

1 (0 mg/kg)

2 (15 mg/kg)

3 (50 mg/kg)

4 (150 mg/kg)

 

No.

%

No.

%

No.

%

No.

%

Females on study

22

 

22

 

22

 

22

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

0

0.0

Females that died

·     Females that aborted

·     Nongravid

·     Gravid

0

0

0

0

0.0

0.0

0.0

0.0

1

0

0

1

4.5

0.0

0.0

100.0

0

0

0

0

0.0

0.0

0.0

0.0

0

0

0

0

0.0

0.0

0.0

0.0

Females that were euthanized

·     Nongravid

·     Gravid

0

0

0

0.0

0.0

0.0

0

0

0

0.0

0.0

0.0

0

0

0

0.0

0.0

0.0

1

0

1

4.5

0.0

100.0

Females examined at scheduled necropsy

·     Nongravid

·     Gravid

o With resorptions only

o With viable fetuses

22

0

22

0

22

100.0

0.0

100.0

0.0

100.0

21

2

19

0

19

95.5

9.5

90.5

0.0

100.0

22

2

20

0

20

100.0

9.1

90.9

0.0

100.0

21

1

20

0

20

95.5

4.8

95.2

0.0

100.0

Total females gravid

22

100.0

20

90.9

20

90.9

21

95.5

Conclusions:
The high dose of 150 mg/kg bw/day was chosen as the effects observed at 300 mg/kg/day in the dose range finder in pregnant rabbits were considered too severe, in particular during the first days of treatment (mean body weight loss of 4% together with severely reduced/almost absent relative food intake over Days 6-9 post-coitum), without a complete recovery at the end of the treatment period as body weight gain and food consumption remained lower than control values during the entire study period.
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as being 150 mg/kg/day.
Executive summary:

The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.The dose levels in this study were selected to be 0, 15, 50 and 150 mg/kg/day, based on the results of the dose range finder.

Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability over 6 hours.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, uterus weight and uterine contents.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in Arachis oil were prepared accurately and homogenously, and were stable over at least 6 hours at room temperature under normal light laboratory conditions.

No maternal toxicity was observedin the 15, 50 and 150 mg/kg/day groups.

No treatment-related changes were noted in any of the maternal parameters investigated in this study (i.e. clinical appearance, body weight, food consumption and macroscopic examination).

For individual animals of all groups, including controls, food consumption was moderately up to severely reduced over several intervals during the treatment period which could be attributed to the oily vehicle used in the current study. This reduction in food consumption was accompanied by slight/mild body weight loss at different intervals in several animals across all groups, including controls, without a clear dose-related response, resulting in one female at 150 mg/kg/day being sacrificed for animal welfare reasons on Day 23 post-coitum due to persistent body weight loss from start of dosing. As this preterm decedent occurred within the normal limits for rabbits of this strain and age housed in the same conditions as in this study, and as the effects in food consumption and body weight were observed across all groups, including controls, without a clear dose-related response, these findings were considered to be unrelated to treatment with the test item.

No developmental toxicity was observedin the 15, 50 and 150 mg/kg/day groups.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as being 150 mg/kg/day. The high dose of 150 mg/kg bw/day was selected as the effects observed at 300 mg/kg/day in the dose range finder in pregnant rabbits were considered too severe, in particular during the first days of treatment (mean body weight loss of 4% together with severely reduced/almost absent relative food intake over Days 6-9 post-coitum), without a complete recovery at the end of the treatment period as body weight gain and food consumption remained lower than control values during the entire study period.

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:
rabbit
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

Dose range finding study

The objective of the dose range finder was to select the dose levels of the test substance for a prenatal developmental toxicity study in New Zealand White Rabbits by oral gavage. Three groups of 6 time-mated females were exposed to test item and vehicle (arachis oil) by gavage at dose levels of 0, 100, and 300 mg/kg from Days 6 to 28 post-coitum, inclusive.

No guidelines are applicable as this study was used for dose level selection purposes only.

Throughout the study, animals were observed for general health/mortality and moribundity. Clinical observations were performed, the animals were weighed and food and water consumption were monitored. Examination of hematology, coagulation and clinical biochemistry was performed. Additionally, macroscopic abnormalities and organ weights were recorded.

Overall, maternal toxicity was observed at 100 and 300 mg/kg bw/day with a dose-related response. Based on the results of this dose range finder study, 150 mg/kg bw/day was established as the high dose level for the main teratology study as animals already showed signs of maternal toxicity when treated at 100 mg/kg bw/day: a reduction in mean body weight gain and food consumption when compared with control group was observed at this dose level, in particular during the first week of treatment (Days 6 - 15 post-coitum), which is a critical period of organogenesis. The effects observed at 300 mg/kg bw/day were considered too severe, in particular during the first days of treatment (mean body weight loss of 4 % together with severely reduced / almost absent relative food intake over Days 6 - 9 post-coitum), without a complete recovery at the end of the treatment period as body weight gain and food consumption remained lower than control values during the entire study period. When calculated in grams overall body weight gain during the treatment period was decreased by 34 % at 100 mg/kg bw/day and by 57 % at 300 mg/kg bw/day.

Therefore, selected dose levels for the prenatal developmental toxicity study were 0, 15, 50 and 150 mg/kg bw/day.

Prenatal developmental toxicity

The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, theNo Observed Adverse Effect Levels(NOAELs) for maternal toxicity and developmental toxicity were evaluated.The dose levels in this study were selected to be 0, 15, 50 and 150 mg/kg/day, based on the results of the dose range finder.

Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability over 6 hours.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, uterus weight and uterine contents.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in Arachis oil were prepared accurately and homogenously, and were stable over at least 6 hours at room temperature under normal light laboratory conditions.

No maternal toxicity was observedin the 15, 50 and 150 mg/kg/day groups.

No treatment-related changes were noted in any of the maternal parameters investigated in this study (i.e. clinical appearance, body weight, food consumption and macroscopic examination).

For individual animals of all groups, including controls, food consumption was moderately up to severely reduced over several intervals during the treatment period which could be attributed to the oily vehicle used in the current study. This reduction in food consumption was accompanied by slight/mild body weight loss at different intervals in several animals across all groups, including controls, without a clear dose-related response, resulting in one female at 150 mg/kg/day being sacrificed for animal welfare reasons on Day 23 post-coitum due to persistent body weight loss from start of dosing. As this preterm decedent occurred within the normal limits for rabbits of this strain and age housed in the same conditions as in this study, and as the effects in food consumption and body weight were observed across all groups, including controls, without a clear dose-related response, these findings were considered to be unrelated to treatment with the test item.

No developmental toxicity was observedin the 15, 50 and 150 mg/kg/day groups.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as being 150 mg/kg/day. The high dose of 150 mg/kg bw/day was selected asthe effects observed at 300 mg/kg/day in the dose range finder in pregnant rabbits were considered too severe, in particular during the first days of treatment (mean body weight loss of 4% together with severely reduced/almost absent relative food intake over Days 6-9 post-coitum), without a complete recovery at the end of the treatment period as body weight gain and food consumption remained lower than control values during the entire study period.

In an additional developmental study performed according to OECD guideline 414 and under GLP, pregnant rats received daily dosages of 0 (Control), 100, 300 and 1000 mg/kg/day by gavage from Day 6 to Day 15 of pregnancy inclusive (Huntingdon, 1991). On Day 20 of pregnancy, the females were killed and subjected to post mortem examination. Litter parameters were determined and approximately half the foetuses subsequently examined for visceral, and the remainder for skeletal abnormalities.

Treatment at 1000 mg/kg/day was associated with post-dosing salivation (occasionally brown-stained) and wet coats in all animals on the majority of days during the dosing period, an increase in mean water consumption throughout the dosing period and slightly retarded bodyweight gain to Day 8 of pregnancy followed by recovery after the end of the dosing period.

Treatment at 300 mg/kg/day was associated with post-dosing salivation (occasionally brown-stained) and wet coats in all animals; the majority being affected on at least three days of the dosing period.

Treatment at 100 mg/kg/day was associated with post-dosing salivation (occasionally brown-stained) and/or wet coats were apparent on occasional days during the dosing period. Fewer animals were affected at this dosage than at 1000 or 300 mg/kg/day.

Although there was a marginal increase in the incidence of early embryonic deaths at 1000 mg/kg/day, this finding was considered to be of doubtful toxicological importance, in view of the low number of litters affected, the lack of statistical significance and the absence of an effect on any other litter parameters.

Treatment with tri-isobutylphosphate showed no obvious adverse effect on embryonic and foetal development as assessed by overall incidence and types of malformations, visceral and skeletal anomalies and skeletal variants. A slight increase in bilateral forelimb flexures (associated with distorted rib cage and thickened ribs in most cases) was confined to only two litters and, in the absence of any dosage-dependent pattern for other abnormalities, was considered unlikely to be treatment-related.

 Within the context of this study, oral administration of tri-isobutylphosphate during Days 6 to 15 of pregnancy produced minimal maternal toxicity at 1000 mg/kg/day, manifested as initial retardation of bodyweight gain and also possibly the increased water consumption. Post-dosing salivation was also a treatment-related maternal sign at this dosage level.

With the exception of post-dosing salivation at 300 and 100 mg/kg/day there were no treatment-related effects of either of these dosages.

Treatment of the parent female produced no selective effect on the unborn litter as assessed by litter values and embryofoetal development and morphology.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available oral prenatal developmental toxicity study is reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for reproduction toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.