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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2020 - 16 April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD guideline no. 414 “Prenatal Developmental Toxicity Study” of 25 June 2018
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-(2-butoxyethoxy)ethoxy)methane
EC Number:
205-598-9
EC Name:
Bis(2-(2-butoxyethoxy)ethoxy)methane
Cas Number:
143-29-3
Molecular formula:
C17H36O6
IUPAC Name:
bis(2-(2-butoxyethoxy)ethoxy)methane
Test material form:
liquid
Remarks:
clear
Details on test material:
Identity Bis(2-(2-butoxyethoxy)ethoxy)methane
CAS no. 143-29-3
Batch no. 00975536W0
Retest date 24 December 2020
Storage conditions Room Temperature
ERBC No. 16564
Specific details on test material used for the study:
Source BASF SE
Identity Bis(2-(2-butoxyethoxy)ethoxy)methane
CAS no. 143-29-3
Batch no. 00975536W0
Retest date 24 December 2020
Storage conditions Room temperature
ERBC no. 16564

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia S.r.l., at arrival, the animals were lower than 2.5 kg body weight and less than 13 weeks of age (9-10 weeks of age).
- Age at study initiation: 18-19 weeks
- Weight at study initiation: 3.46-3.53 kg
- Housing: The animals were individually housed in polycarbonate/stainless steel cages with perforated NorylTM floor suspended over trays. Each cage tray held absorbent paper which was inspected and changed as necessary.
- Diet (e.g. ad libitum): A commercially available laboratory rabbit diet (Mucedola 2 RB 15,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum throughout the study.
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: An acclimatisation period of approximately 9 weeks was allowed before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
CMC 0.5% w/v
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle. The formulations were prepared daily or up to weekly interval in agreement with stability data to reach the required concentrations of 3, 10 and 30mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.

Analysis was performed in separate studies in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (RTC study nos.
94840, 94860 and ERBC Study no. A3671). In the present study, the stability at 24 hours at room temperature and for 7 days at nominal +4°C, was verified at 3 mg/mL.

Samples of the formulations prepared during the current study (the first and the last week of treatment, where possible) were analysed to check the homogeneity and concentration.
Chemical analysis was carried out by the Analytical ChemistryDepartment at ERBC. Results of the analyses were within the acceptability limits stated in ERBC SOPs

The test item was administered orally, by gavage, at a dose volume of 5mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the
volume administered was recorded for each animal.

VEHICLE
The vehicle was an aqueous solution of carboxymethylcellulose (CMC 0.5% w/v).
- Justification for use and choice of vehicle (if other than water): common used vehicle
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in separate studies in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (RTC study nos.
94840, 94860 and ERBC Study no. A3671). In the present study, the stability at 24 hours at room temperature and for 7 days at nominal +4°C, was verified at 3 mg/mL.
Samples of the formulations prepared during the current study (the first and the last week of treatment, where possible) were analysed to check the homogeneity and concentration.
Chemical analysis was carried out by the Analytical ChemistryDepartment at ERBC. Results of the analyses were within the acceptability limits stated in ERBC SOPs

Validation of the analytical method
The analytical method was validated in RTC Study no. 94840 in the range from 1 to 100mg/mL using a GC/FID technique with Chromeleon 7.2.9. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.98; accuracy 90-110%; precision CV < 5%).

Preparation sampling
Preparations of the test item were prepared as suspensions in carboxymethylcellulose (CMC 0.5% w/v). Concentration and homogeneity of the low and high dose level were assessed by taking six analytical aliquots in different positions. For the intermediate level, only concentation was assessed by taking two different analytical aliquots. Concentration was evaluated as the mean of the single determinations and homogeneity was evaluated as the coefficient of variation of the sextuplicate.

Stability
In RTC Study no. 94860, a 24 hour stability at room temperature was verified in the range from 4 to 40mg/mL; in ERBC Study no. A3671 a 24 hour stability at room temperature and an 8 days stability at +5°C ± 3°C were verified at 8 and 80mg/mL; in the present study a 24 hour stability at room temperature and a 7 days stability at +5°C ± 3°C were verified at 3mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (85%-115% for concentration and CV < 10% for homogeneity).
Details on mating procedure:
Females will be introduced to sexually mature males obtained from the same supplier. Each female will remain with the male (in the home cage of the male) for at least 1 hour after
successful mating has been observed. The day successful mating is detected will be considered Day 0 post coitum (or gestation Day
0).

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: at least 1 hour

Duration of treatment / exposure:
from Day 6 through Day 28 post coitum
Frequency of treatment:
once a day
Duration of test:
till day 29 post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a preliminary, non-GLP Compliant Study (ERBC Study no. Y0440). In this study, groups of 6 mated rabbits received the test compound at dose levels of 0, 50 or 200 mg/kg/day. Ataxia was recorded in all females at the high dose level of 200 mg/kg/day. Convulsions semi-closed eyes and distended hindlimbs were also recorded in single occasions at this dosage. No signs were observed at the low dose level of 50 mg/kg/day. Therefore, the high dose level of 150 mg/kg/day was selected for the present study.

- Rationale for animal assignment (if not random): The rabbits were allocated to the groups by stratified randomisation to give approximately equal initial group mean body weights.

Examinations

Maternal examinations:
As soon as possible before start of mating, each animal was given a detailed physical examination by a veterinarian. Particular attention was paid to external genitalia and
mammary glands. These data are not presented in the present report but retained as study raw data.

CAGE SIDE OBSERVATIONS: Yes

- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
All clinical signs were recorded for individual animals. The animals were observed once fromgestationDay 0 up toDay 5. During the treatment period additional sessions of clinical
signs were included at the same time interval each day. The interval was selected taking into consideration the presence of post-dose reactions.
The observation of females was carried out approximately at the following intervals:
– session 1 = before dosing
– session 2 = 5-15 minutes after dosing
– session 3 = 1.5 - 2 hours

BODY WEIGHT: Yes
Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on Days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post coitum.

FOOD CONSUMPTION : Yes
Food consumption was recorded on Days 3, 6, 9, 12, 15, 18, 24, 27 and 29 of post coitum starting from Day 0 post coitum.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
The animals that completed the scheduled test period, were killed with intravenous injection of a suitable euthanasia agent (Tanax®.Tanax was injected in sedated animals with
Medetomidine hydrochloride) and subjected to necropsy
All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental.

Necropsy
All animals, including those killed for humane reasons, were subjected to necropsy and
the number of implantations and corpora lutea were recorded. The clinical history of the
animal was studied and a detailed post mortem examination was conducted (including examination
of the external surface and orifices). Changes were noted and the abnormalities
preserved in 10% neutral buffered formalin.
The ovaries and uteri were examined to determine:
– Gravid uterine weight (not obtained from animals killed during the study);
– Corrected maternal body weight (terminal body weight minus gravid uterus weight)
– Corrected maternal body weight gain (corrected maternal body weight minus maternal
body weight on gestation Day 3)
– number of corpora lutea for pregnant animals;
– number of implantations for pregnant animals;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements
and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.
Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.
Uteri or individual uterine horns without visible implantations were immersed in a 20%
solution of ammonium sulphide to reveal evidence of embryonic death at very early stages
of implantation.

OTHER:
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
– Gravid uterine weight (not obtained from animals killed during the study);
– Corrected maternal body weight (terminal body weight minus gravid uterus weight)
– Corrected maternal body weight gain (corrected maternal body weight minus maternal
body weight on gestation Day 3)
– number of corpora lutea for pregnant animals;
– number of implantations for pregnant animals;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements
and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.
Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.
Uteri or individual uterine horns without visible implantations were immersed in a 20%
solution of ammonium sulphide to reveal evidence of embryonic death at very early stages
of implantation.
Fetal examinations:
All viable foetuses were euthanised as described above, weighed and examined externally and internally. The thoracic and abdominal cavities were opened and examined
and sex was determined. Head section (medial suture bones) was performed at necropsy on each foetus, selected for skeletal examination, and particular attention was paid to the brain ventricles. The head from approximately half of the foetuses (i.e. routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed examination of internal structures of the head: eye, brain, nasal passages and tongue. Foetuses were eviscerated, skinned and fixed in 95% ethanol for subsequent skeletal staining and examination. Skeletal and fixed head examinations were performed in all groups.

Structural deviations were classified as follows:

Malformations
Major abnormalities that are rare and/or affect the survival or health of the species underinvestigation.

Anomalies
Minor abnormalities that are detected relatively frequently.

Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis
that would have otherwise followed a normal pattern of development.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal- Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of theWilliams test. The criterion for statistical significance was p<0.05.
Indices:
Group mean values for body weight and food consumption of pregnant females, gravid uterus weight, corrected maternal body weight, corrected maternal body weight gain, litter size, intra-uterine deaths, corpora lutea count, total implantation loss and pre- and postimplantation loss were calculated. Data from non-pregnant animals were not included in group mean calculations. The animal which aborted was included in body weight and food consumption calculations up to the day of abortion.

Litter data
Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss%=
no. of corpora lutea−no. of implantations/
no. of corpora lutea
×100
Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss%=
no. of implantations−no. of live foetuses/
no. of implantations
×100
Sex ratios of the foetuses were calculated as the percentage of males. The number of foetuses affected with structural deviations and the corresponding litter percentage were calculated. All derived values (e.g. means, percentages, ratios) were first calculated within the litter and the group values derived as a mean and standard deviation of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ataxia, fromslight to marked severity, was recorded in all animals at 150mg/kg/day throughout the treatment period. One female at 150 mg/kg/day (Dam no. 177) had one episode of convulsion on the first day of treatment (Day 6 post coitum) followed by lateral decubitus.
The same animal showed bradypnoea on gestation Day 10.
Tremors of slight to moderate severity were seen on single occasion at 150 mg/kg/day (Dam no. 167) whereas decreased activity was recorded in most of females at 150 mg/kg/day. All these signs occurred between 5 and 15 minutes after dosing andwere not longer detected approximately 2 hours post dose, indicating a complete recovery.Signs of hairloss, damaged ear or reduced faeces were recorded in single animals regardless the treatment groups and considered unrelated to treatment.
No treatment-related clinical signs were recorded at 15 and 50 mg/kg/day. The abortion (Dam no. 113) that occurred at 50 mg/kg/ day was considered of incidental nature.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two premature deaths occurred in the study, one female at 15 mg/kg/day sacrificed due to the presence of severe clinical signs, coherent with a mis-dosing and one female at 50 mg/kg/day which was sacrificed following abortion.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect on body weight and body weight gain throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not affected by treatment.
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:
no effects observed
Description (incidence and severity):
No statistically significantly differences were seen in the uterus weight, corrected maternal body weight (terminal body weight minus the uterus weight) and maternal corrected body weight gain, calculated subtracting the uterus weight and the body weight at gestation Day 3 from the terminal body weight, between treated groups and control groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination
Unscheduled deaths
One female at 15 mg/kg/day was sacrificed following dyspnoea, cyanotic appearance and the gross observations were consisting with a mis-dosing. One female at 50 mg/kg/day was sacrificed following abortion.
Final sacrifice
Animals euthanized at termination did not show any macroscopic changes that could be considered treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 50 mg/kg/day (Dam no. 113) was sacrificed following abortion on gestation Day 25. No signswere recorded up to the day of abortion. The cause of this loss of pregnancy was not attributed to the test item as the abortion occurred at low incidence and in the absence of other signs of concern.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
All reproductive outcomes, including the number of corpora lutea, number of implantations, implantation loss, intrauterine deaths and number of live foetuses were unaffected by treatment. The mean litter weight and foetal weight of both sexes were also comparable between treated and control groups.
No differences were seen in the sex ratios.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 9 females were found not pregnant at final sacrifice. The majority were found in the high dose group (150 mg/kg/day). This occurrence appeared to be unrelated to the mating performance of males since the same male mated different females among the groups and induced pregnancy. Moreover, the mating was performed in batches and females were randomly allocated across the groups. Finally, the male rabbits
were also evenly distributed for copulation avoiding to allocate females mated with the same males in the same group, where possible. Thus, this occurrence was considered as incidental.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean litter weight and foetal weight of both sexes were also comparable between treated and control groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All reproductive outcomes, including the number of corpora lutea, number of implantations, implantation loss, intrauterine deaths and number of live foetuses were unaffected by treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No differences were seen in the sex ratios.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter weight and foetal weight of both sexes were also comparable between treated and control groups.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
All reproductive outcomes, including the number of corpora lutea, number of implantations, implantation loss, intrauterine deaths and number of live foetuses were unaffected by treatment.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
The skeletal examination did not reveal treatment-related findings.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Slight enlarged brain ventricles were found in one single foetus at 50 (foetus no. 3; Dam no. 119) and one at 150 mg/kg/day (foetus no. 1; Dam no. 111). These findings occurred at very low incidence, did not follow a dose dependency and they were considered unrelated to treatment.
Foetuses small in size (body weight lower than 35g) were seen in all groups without substantial differences.
No other findings were recorded.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No signs of developmental toxicity were seen in all groups.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Maternal toxicity
Signs of toxicity were confined to the transient presence of ataxia and decreased activity at 150 mg/kg/day. Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity is 50 mg/kg/day.
Developmental toxicity
No signs of developmental toxicity were seen in all groups. Based on these results, the NOAEL (No Observed Adverse Effect Level) for developmental toxicity is 150 mg/kg/day.
Executive summary:

The effects of Bis(2-(2-butoxyethoxy)ethoxy)methane were investigated in New Zealand White rabbits during pregnancy and embryo-foetal development according to OECD 422 guideline (BASF SE 2021).

Females were in-house mated with sexually mature males of the same strain, held as ERBC stock animals, and then assigned to 4 groups of 24 animals and treated with test item as supplied. Females were administered by oral gavage during the gestation period from Day 6 through

Day 28 post coitum at the dose levels of 15, 50 and 150mg/kg/day. The dose volume was set at 5mL/kg body weight. Females from the control group (Group 1) received 0.5% carboxymethylcellulose (0.5% CMC) as vehicle at the same dose volume during the same treatment period. Mortality check, clinical signs, body weight and food consumption were recorded during the in-life phase. At completion of the study period, females were caesarean-sectioned on Day 29 post coitum and subjected to detailed post mortem examination. The gravid uterus was weighed and the uterine content examined for the number of implantations, intra-uterine deaths and live foetuses. Live foetuses were weighed, sexed and observed for external and internal abnormalities. The ovaries were also examined and the corpora lutea counted. Examination of soft fixed head and the alizarin stained skeleton was performed in foetuses from all groups.

Two premature deaths occurred in the study, one female at 15 mg/kg/day sacrificed due to the presence of severe clinical signs, coherent with a mis-dosing and one female at 50 mg/kg/day which was sacrificed following abortion. The number of females with viable foetuses on gestation Day 29 was: 24, 21, 22 and 18 in the control, 15, 50 and 150 mg/kg/day, respectively.

The recurrent clinical signs were ataxia and decreased activity at 150 mg/kg/day. These signs were transient and the animals then recovered.

No treatment-related clinical signs were seen at 15 and 50 mg/kg/day. There was no effect on body weight and body weight gain throughout the study and the food consumption was not affected by treatment. No statistically significantly differences were seen in the uterus weight, corrected maternal body weight (terminal body weight minus the uterus weight) and maternal corrected body weight gain, calculated subtracting the uterus weight and the body weight at gestation Day 3 from the terminal body weight, between treated groups and control groups. One female at 15 mg/kg/day which was sacrificed following dyspnoea, cyanotic appearance and the gross observations were consisting with a mis-dosing. One female at 50 mg/kg/day was sacrificed following abortion. Animals euthanized at termination did not show any macroscopic changes that could be considered treatment-related. Litter data and sex ratio were unaffected by treatment and no treatment-related findings were seen at external and internal examination of foetuses. Soft fixed head examination did not show treatment-related changes and the skeletal examination did also not reveal treatment-related findings.

Concluding signs of maternal toxicity were confined to the transient presence of ataxia and decreased activity at 150 mg/kg/day. These effects did not affect the pregnancy outcome but they were considered adverse. Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity is 50 mg/kg/day.

Further no signs of developmental toxicity were seen in all groups. Based on these results, the NOAEL (No Observed Adverse Effect Level) for developmental toxicity is 150 mg/kg/day.