Bis(2-(2-butoxyethoxy)ethoxy)methane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Rat, Combined repeated dose toxicity/ Reproduction toxicity screenig study: NOAEL parental toxicity = 100 mg/kg bw due to decreased body weight, food consumption, increased liver weights and microscopic liver changes in the higher dose; NOAEL fertility = 100 mg/kg bw due to decreased fertility indices and increased pre-birth loss but with large dose spacing (next level 800mg/kg)(GLP, OECD 422, RTC/ Rohm & Haas 2004)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study, however spacing of doses is not adequate and historical control data is missing

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on mating procedure:

Pairings were monogamous. Vaginal smears were taken during pairing up to the day of positive identification of mating. Each cage was checked each morning for the presence of copulation plugs. The pairing combinations of animals which had not had positive identification of mating after 14 days of pairing were changed within each treatment group. Animals that did not have signs of mating during the initial pairing were placed with a second male that previously had positive signs of mating. The subsequent pairing was monitored for mating as described above for the first pairing. No more than 28 days was allowed for pairing.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Exposure period: at least ca. 6 weeks:
For males, treatment commenced when the males were approximately 12 weeks old and continued for two weeks prior to pairing, during pairing until mating was confirmed, and up to and including the day before sacrifice.
For females, treatment commenced when they were approximately 12 weeks old and continued for two weeks prior to pairing through the mating and gestation periods, up to day 3 of lactation. Dams and offspring were sacrificed on Day 4 post-partum.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
10, 100, 800 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

see information in IUCLID section 7.5.1. RTC (2004).

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning, starting two weeks before pairing (first day of dosing) until a positive identification of mating was made. The vaginal smear data were examined to determine anomalies of the estrous cycle, and the pre-coital interval (i.e., the number of nights paired prior to the detection of mating.

Sperm parameters (parental animals):

Each cage was checked each morning for the presence of copulation plugs.

Litter observations:

The total litter size (live and dead) was counted as soon as possible after parturition. Live pups were identified individually within the litter by toe amputation, sexed and examined for external abnormalities. Live and dead pups were counted and weighed on days 1 and 4 post-partum, with the exception of one mid-dose female, where pups were weighed on days 2 and 5 post-partum. All litters were observed daily. All pups found dead were given a post-partum examination.

Postmortem examinations (parental animals):

- Organ weights P: The following organs from all adult animals in all groups were dissected free of fat and weighed: adrenals, brain, epididymides, heart, kidneys, liver, spleen, thymus, testes.

- Histopathology P: The tissues listed, as follows, were examined in all adult animals: corpora abnormalities (gross lesions), adrenals (paired), bone marrow (from the sternum), brain, caecum, coagulating glands (paired), colon, duodenum, epididymides (paired), esophagus, heart, ileum, jejunum, kidneys (paired), liver, lymph nodes (cervical), lymph nodes (mesenteric), lungs, ovaries with oviduct (paired), prostate, pituitary, rectum, sciatic nerve, seminal spinal column with spinal cord, spleen, stomach, thymus, thyroid, trachea, testes (paired), urinary bladder, uterus with cervix, vagina.

Statistics:

For continuous variables, the significance of the difference amongst group means was assessed by analysis of variance. Differences between the treated group and the control group were assessed by Dunnett's test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett's Test before Dunnett's Test was performed. If the data were found to be inhomogeneous, a modified t-test (Cochran and Cox) was applied. Statistical analysis of histopathological findings was not carried out by means of the non-parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for all other parameters. Intergroup differences between the control and the treated group were assessed by the non-parametric version of the Williams' test.

Body weight:
Body weight and body weight gain were unaffected by treatment in the males throughout the study and in females before pairing. Slight decreases, not statistically significant, in body weight and body weight gain were observed in the high dose females during the gestation period when compared to controls. No differences were noted in females of any treatment group during the post-partum period when compared to controls.

Food consumption:
Food consumption remained comparable between control and treated groups in both sexes before pairing. A statistically significant reduction in food consumption was noted in high dose females on day 7 post-coitum. A similar reduction (not statistically significant) was noted on day 14 post-coitum in the same group.

Description, severity, time of onset and duration of clinical signs:
Weekly physical examinations including detailed clinical signs with neurotoxicity assessment did not show any signs which could be correlated to the treatment with the test item in males or females in any group during the treatment period.

Fertility index:
Fertility index in the high-dose group was decreased compared to controls. A treatment-related effect on fertility was apparent in the high dose group. A total of fifteen females proved not to be pregnant at necropsy; three in the control group and in the mid-dose group, two in the low-dose group and seven in the high-dose group.

Pre-coital interval:
Pre-coital interval in tested rats was comparable to controls.

Duration of gestation:
Gestation periods in tested rats were comparable to controls.

Gestation index:
An increased incidence, not statistically significant, in the number and percentage of pre-birth loss was noted in high dose females when compared to controls.

Changes in lactation: not characterized

Changes in estrus cycles:
The estrus cycles of tested animals were comparable to controls.

Effects on sperm: not characterized

Hematological findings incidence and severity:
No toxicologically significant changes in hematology occurred in this study. Statistically significant decreases in prothrombin time were observed in the high dose male group. This change was considered incidental and not treatment-related since it was seen in only one sex, and the magnitude of the effect was low.
Statistically significant decreases in neutrophil percentage were observed in the high dose females. This change was considered incidental and not treatment-related since it was seen in one sex only and no other changes were seen in the white blood cell differential counts.

Clinical biochemistry findings incidence and severity:
No toxicologically significant change in any clinical chemistry parameters occurred at any dose level.
A statistically significant decrease in alkaline phosphatase level was observed in the high dose male group. Since this change was minimal, was decreased compared to the control group and occurred in only one sex, it was not considered toxicologically significant. A statistically significant decrease in aminotransferase level was noted in the low dose male group when compared to controls. This decrease was considered incidental to treatment since no dose response was evident.
Statistically significant decreases in urea were seen in the low and high dose males compared to controls. These changes were not considered treatment-related since the changes were decreased relative to controls and there was no evident dose response. Statistically significant changes in creatinine and inorganic phosphorus levels were observed in the high dose male group. These changes were not considered treatment-related since the values were decreased compared to controls, the magnitude of the change was small and they occurred only in one sex.
Urea level was also statistically significantly decreased in the low dose male group. This change was not considered treatment-related since the effect was decreased, there was no obvious graded dose response at the mid-and high dose groups and the effect occurred in only one sex.
No differences in clinical chemistry parameters were noted between control and treated females.

Mortality:
One mid-dose female was sacrificed, on day 0 post-partum, for humane reasons, since signs of difficult delivery were noted. No other animals died prior to study termination.

Gross pathology incidence and severity:
No macroscopic change was reported at necropsy in the examined organs/tissues of the animals killed at termination that could be considered related to the administration of the test item. The recorded changes were considered to be incidental or spontaneous in origin.

Number of implantations:
There were no effects on the mean number of implantation sites at any dose level.

Number of corpora lutea: not reported

Organ weight changes:
Treatment-related organ weight changes occurred in the liver of high dose males and females. Statistically significant increases in absolute and relative liver weights were observed in the high dose males when compared to controls. Increases in absolute (not statistically significant) and relative (statistically significant) liver weights were also noted in only the high dose females.
Statistically significant increases in absolute and relative kidney weights were noted in the mid-dose male group only.
Since the absolute and relative kidney weights of the high dose males were not affected (i.e., no apparent dose response), there was no corresponding increase in females, and no corresponding microscopic change in this tissue, the kidney weight changes were considered not related to treatment with the test item. Slight, but statistically significant increases in absolute and relative adrenal weights were observed in the high dose female group. This change was considered incidental and not treatment-related since there was no corresponding microscopic histopathologic change in this tissue and similar effects were not seen in the high dose male group.

Histopathology incidence and severity:
No microscopic changes clearly related to test substance administration were observed in male or female rats given 10 or 100 mk/kg bw/day of the test article.
Diffuse hepatocellular hypertrophy (enlarged hepatocytes in all areas of the lobules) occurred in male and female rats of the high dosage group and was considered to be treatment-related.
Focal germ-cell depletion and degeneration occurred in one testis (unilateral) of each of four different male rats of the high dosage group. Exfoliated spermatogenic cells were observed in the epididymis in three of these high dose rats and in one additional high dose rat without testicular changes. The significance of this finding was uncertain. Systemic toxicity usually does not affect only one of a paired organ.
All other microscopic changes were considered to have occurred spontaneously and to be unrelated to test article administration. These changes generally occurred at single, low, or similar frequencies among the groups and their type, incidence or severity was not influenced by compound administration.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Litter size and weights:
A decrease in the average number of pups per litter was observed at birth, and on day 4 post-partum in the high-dose group. Decreased litter weight and mean pup weight in the high-dose group (not statistically significant) were noted at birth and on day 4 post-partum.

Sex and sex ratios:
Sex ratios at birth and on day 4 post-partum did not show any differences between control and treated groups when calculated as the percentage of males.

Viability index:
Pup survival to day 4 was decreased in the high-dose-group. The number of females with live pups on day 4 post-partum was 6, 8, 6, and 3 in the control, low-, mid- and high-dose groups, respectively. A statistically significant increase in pup mortality (females and total) from day 0 to day 4 was noted in the high dose group. Similar results were also seen in male pups but the difference was not statistically significant.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: decreased fertility indices and increased pre-birth loss

Reproductive effects observed:

not specified

The relevance of the observed effects (decreased fertility indices and increased pre-birth losses,

embryo-fetal toxicity as well as developmental effects of microdactyly and/or agenesis of digits (one litter) and

flexure of hind limbs (one litter)) with regard to the hazard assessment

(i.e., classification) is unclear due to the following concerns about the study:

 Low fertility rate for the control group which limits the predictive (i.e., statistical)

power of the study as well as its reliability

 Lack of a clear dose-response relationship (effects only in the high dose group);

Observed parental toxicity raises the concern that 800 mg/kg for the high dose level

may have been too high

 Inadequate sample size for evaluation of fetal malformations in the high dose

group

 Lack of the historical control data in the study report does not allow for

distinguishing between substance-related and spontaneous effects

 Some incorrect values stated in the final report call into question the QA process

for the study, the data collection software, and /or perhaps some of the results

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

oral

A GLP conform repeated dose/ reproductive toxicity study was performed with TP-90B RUBBER CHEMICAL according to OECD 422 (RTC/ Rohm & Haas 2004). Groups of 10 Sprague-Dawley rats per sex received TP-90B Rubber Chemical in 0.5% aqueous carboxymethylcellulose at doses of 10, 100 and 800 mg/kg bw/d by gavage. Males were treated for 2 weeks prior to pairing, through pairing until the day before sacrifice. Females were treated for 2 weeks prior to pairing, through the mating and gestation periods up to day 3 of lactation. Dams and offspring were sacrificed on Day 4 post-partum (= at least 43 days).

Following effects on parental toxicity were observed:

 

Fate of females

One mid dose female was sacrificed on Day 0 post-partum for human reasons since signs of dystocia (difficult delivery) were noted.

 

Weekly clinical signs and neurotoxicity assessment and daily pre- and post-dose observations

Weekly physical examinations including detailed clinical signs and neurotoxicity assessment (Functional Observation Battery) did not show any treatment-related effects.

Daily post-dose observations of clinical signs showed high dose male animals with ataxia,

Semiclosed eyes, hunched posture, reduced activity and salivation. Pronation, ataxia, semiclosed eyes, reduced activity, hunched posture, lethargy, twitches, piloerection and salivation were noted at post-dose observations in high dose females.

 

Body weight and body weight gain

Slight decreases in body weight and body weight gain occurred only in high dose females during gestation.

 

Food consumption

Food consumption remained comparable between control and treated groups in both sexes before pairing. Reduced food consumption was noted in high dose females on Days 7 and 14 post-coitum.

 

Motor activity and sensory reaction to stimuli

Motor activity measurements and sensory reaction to stimuli were unaffected by treatment at any dose level in both sexes.

 

Haematology

No toxicologically significant change in haematology values occurred in males or females at any dose level.

 

Clinical chemistry

No toxicologically significant change in any clinical chemistry parameter occurred in males or females at any dose level.

 

Terminal body weight and organ weights

No differences in terminal body weights were observed between treated and control animals.

Absolute and relative liver weights were increased in high dose males and females when compared to controls. All changes were statistically significantly different from controls except the absolute liver weight in females.

 

Macroscopic examination

No macroscopic change was reported at necropsy in the examined organs/tissues of the animals killed at termination that could be considered related to the administration of the test item. The gross changes observed were considered to be incidental or spontaneous in origin.

 

Microscopic examination

No microscopic changes clearly related to test item administration were observed in male or female rats in the low and mid-dose groups. Diffuse hepatocellular hypertrophy (enlarged hepatocytes in all areas of the lobules) occurred in male and female rats of the high dose group. A finding of uncertain significance was observed in the high dose group.

Focal germ cell depletion and degeneration in one testis (unilateral) occurred in four high dose male rats. Three of these four rats also had a few exfoliated spermatogenic cells in the lumen of the epididymal tubules.

 

 

Following effects on fertility were observed:

Reproductive parameters

Oestrus cycle, copulation plug, pre-coital interval and copulatory index at all dose levels were comparable to controls. Fertility index in the high dose group was decreased compared to control: Thirty-nine females in the study showed positive signs of mating. Fifteen females proved not to be pregnant at necropsy; three in the control and in the mid-dose group, two in the low dose group and seven in the high dose group. In addition, two control and one mid-dose female showed unilateral implantation. One low dose female did not mate. The number of females with live pups on Day 4 post-partum was 6, 8, 6, and 3 in the control, low, mid and high dose groups, respectively.

 

Implantation and pre-birth loss

Gestation periods were similar between control and treated groups. All dams gave birth on Day 22 or Day 23 post-coitum. The number and percentage of pre-birth loss of pups in the high dose females was increased compared to controls.

 

Litter data and sex ratios

Cumulative pup loss was statistically significantly increased in the high dose group on Day 4 post-partum. Litter weight and mean pup weight in the high dose group were lower than controls at birth and on Day 4 post-partum. A decrease in the average number of pups per litter was observed at birth and on Day 4 post-partum in the high dose group. Pup survival to Day 4 post-partum was decreased in the high dose group. Increased pup mortality from Day 0 to Day 4 was noted in the high dose group. Sex ratios at birth and on Day 4 post-partum did not show any differences between control and treated groups.

 

 

Based on the results of the study, the NOAEL for maternal toxicity is considered to be 100 mg/kg bw/day due to decreased body weight, food consumption, increased liver weights and microscopic liver changes; the NOAEL for effects on reproduction is considered to be 100 mg/kg be/day due to decreased fertility indices and embryo-fetal toxicity. However, a low fertility rate was observed also in the control group and historical control data useful for assessment are missing in the report. Additionally, adverse effects were observed only in the highest dose without a clear dose relationship. In conclusion, a final assessment of the observed effects cannot be made.

 

Summary of the concerns with the reliability of this study:

The relevance of the observed effects (decreased fertility indices and increased pre-birth losses,

embryo-fetal toxicity as well as developmental effects of microdactyly and/or agenesis of digits (one litter) and

flexure of hind limbs (one litter)) with regard to the hazard assessment

(i.e., classification) is unclear due to the following concerns about the study:

 Low fertility rate for the control group which limits the predictive (i.e., statistical)

power of the study as well as its reliability

 Lack of a clear dose-response relationship (effects only in the high dose group);

Observed parental toxicity raises the concern that 800 mg/kg for the high dose level

may have been too high

 Inadequate sample size for evaluation of fetal malformations in the high dose

group

 Lack of the historical control data in the study report does not allow for

distinguishing between substance-related and spontaneous effects

 Some incorrect values stated in the final report call into question the QA process

for the study, the data collection software, and /or perhaps some of the results

Short description of key information:
oral
Rat, Combined repeated dose toxicity/ Reproduction toxicity screenig study: NOAEL parental toxicity = 100 mg/kg bw due to decreased body weight, food consumption, increased liver weights and microscopic liver changes in the higher dose; NOAEL fertility = 100 mg/kg bw due to decreased fertility indices and increased pre-birth loss (GLP, OECD 422, RTC/ Rohm & Haas 2004)

Effects on developmental toxicity

Description of key information

OECD 414, rat (RTC, 2013):  NOAEL maternal = 120mg/kg bw/d, NOAEL developmental = 400mg/kg bw/d
OECD 414, rabbit (BASF SE 2021): NOAEL maternal = 50mg/kg bw/d, NOAEL developmental = 150mg/kg bw/d

Link to relevant study records

Reference

Reference 1

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

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)

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

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

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

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.

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.

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

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.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

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

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.

Key result

Developmental effects observed:

no

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study, however spacing of doses is not adequate and historical control data is missing

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on mating procedure:

Pairings were monogamous. Vaginal smears were taken during pairing up to the day of positive identification of mating. Each cage was checked each morning for the presence of copulation plugs. The pairing combinations of animals which had not had positive identification of mating after 14 days of pairing were changed within each treatment group. Animals that did not have signs of mating during the initial pairing were placed with a second male that previously had positive signs of mating. The subsequent pairing was monitored for mating as described above for the first pairing. No more than 28 days was allowed for pairing.

Duration of treatment / exposure:

Duration of test: at least ca. 6 weeks
For males, treatment commenced when the males were approximately 12 weeks old and continued for two weeks prior to pairing, during pairing until mating was confirmed, and up to and including the day before sacrifice.
For females, treatment commenced when they were approximately 12 weeks old and continued for two weeks prior to pairing through the mating and gestation periods, up to day 3 of lactation. Dams and offspring were sacrificed on Day 4 post-partum.

Frequency of treatment:

TP-90B was administered to rats daily

Remarks:

Doses / Concentrations:
10, 100, 800 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Maternal examinations:

see information in IUCLID section 7.5.1. RTC (2004).

Fetal examinations:

The total litter size (live and dead) was counted as soon as possible after parturition. Live pups were identified individually within the litter by toe amputation, sexed and examined for external abnormalities. Live and dead pups were counted and weighed on days 1 and 4 post-partum, with the exception of one mid-dose female, where pups were weighed on days 2 and 5 post-partum. All litters were observed daily. All pups found dead were given a post-partum examination.

Statistics:

For continuous variables, the significance of the difference amongst group means was assessed by analysis of variance. Differences between the treated group and the control group were assessed by Dunnett's test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett's Test before Dunnett's Test was performed. If the data were found to be inhomogeneous, a modified t-test (Cochran and Cox) was applied. Statistical analysis of histopathological findings was not carried out by means of the non-parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for all other parameters. Intergroup differences between the control and the treated group were assessed by the non-parametric version of the Williams' test.

Historical control data:

not available in the study report

Details on maternal toxic effects:

Details on maternal toxic effects:
- Mortality and day of death: One mid-dose female was sacrificed, on day 0 post-partum, for humane reasons, since signs of difficult delivery were noted. No other animals died prior to study termination.

- Number pregnant per dose level: A treatment-related effect on fertility was apparent in the high dose group. A total of fifteen females proved not to be pregnant at necropsy; three in the control group and in the mid-dose group, two in the low-dose group and seven in the high-dose group.

- Number aborting: A decrease in the average number of pups per litter was observed at birth in the high-dose group.

- Number of resorptions: An increased incidence, not statistically significant, in the number and percentage of pre-birth loss was noted in high dose females when compared to controls.

- Number of implantations: There were no effects on the mean number of implantation sites at any dose level.

- Number of corpora lutea: not reported

- Duration of Pregnancy: Gestation periods were similar between controls and tested groups.

- Body weight: Body weight and body weight gain were unaffected by treatment in the males throughout the study and in females before pairing. Slight decreases, not statistically significant, in body weight and body weight gain were observed in the high dose females during the gestation period when compared to controls. No differences were noted in females of any treatment group during the post-partum period when compared to controls.

- Food consumption: Food consumption remained comparable between control and treated groups in both sexes before pairing. A statistically significant reduction in food consumption was noted in high dose females on day 7 post-coitum. A similar reduction (not statistically significant) was noted on day 14 post-coitum in the same group.

- Description, severity, time of onset and duration of clinical signs: Weekly physical examinations including detailed clinical signs with neurotoxicity assessment did not show any signs which could be correlated to the treatment with the test item in males or females in any group during the treatment period.

- Hematological findings incidence and severity: No toxicologically significant changes in hematology occurred in this study. Statistically significant decreases in prothrombin time were observed in the high dose male group. This change was considered incidental and not treatment-related since it was seen in only one sex, and the magnitude of the effect was low.
Statistically significant decreases in neutrophil percentage were observed in the high dose females. This change was considered incidental and not treatment-related since it was seen in one sex only and no other changes were seen in the white blood cell differential counts.

- Clinical biochemistry findings incidence and severity: No toxicologically significant change in any clinical chemistry parameter occurred at any dose level.
A statistically significant decrease in alkaline phosphatase level was observed in the high dose male group. Since this change was minimal, was decreased compared to the control group and occurred in only one sex, it was not considered toxicologically significant. A statistically significant decrease in aminotransferase level was noted in the low dose male group when compared to controls. This decrease was considered incidental to treatment since no dose response was evident.
Statistically significant decreases in urea were seen in the low and high dose males compared to controls. These changes were not considered treatment-related since the changes were decreased relative to controls and there was no evident dose response. Statistically significant changes in creatinine and inorganic phosphorus levels were observed in the high dose male group. These changes were not considered treatment-related since the values were decreased compared to controls, the magnitude of the change was small and they occurred only in one sex.
Urea level was also statistically significantly decreased in the low dose male group. This change was not considered treatment-related since the effect was decreased, there was no obvious graded dose response at the mid-and high dose groups and the effect occurred in only one sex.
No differences in clinical chemistry parameters were noted between control and treated females.

- Gross pathology incidence and severity: No macroscopic change was reported at necropsy in the examined organs/tissues of the animals killed at termination that could be considered related to the administration of the test item. The recorded changes were considered to be incidental or spontaneous in origin.

- Organ weight changes: Treatment-related organ weight changes occurred in the liver of high dose males and females. Statistically significant increases in absolute and relative liver weights were observed in the high dose males when compared to controls. Increases in absolute (not statistically significant) and relative (statistically significant) liver weights were also noted in only the high dose females.
Statistically significant increases in absolute and relative kidney weights were noted in the mid-dose male group only. Since the absolute and relative kidney weights of the high dose males were not affected (i.e., no apparent dose response), there was no corresponding increase in females, and no corresponding microscopic change in this tissue, the kidney weight changes were considered not related to treatment with the test item. Slight, but statistically significant increases in absolute and relative adrenal weights were observed in the high dose female group. This change was considered incidental and not treatment-related since there was no corresponding microscopic histopathologic change in this tissue and similar effects were not seen in the high dose male group.

- Histopathology incidence and severity: No microscopic changes clearly related to test substance administration were observed in male or female rats given 10 or 100 mg/kg/day of the test article.
Diffuse hepatocellular hypertrophy (enlarged hepatocytes in all areas of the lobules) occurred in male and female rats of the high dosage group and was considered to be treatment-related.
Focal germ-cell depletion and degeneration occurred in one testis (unilateral) of each of four different male rats of the high dosage group. Exfoliated spermatogenic cells were observed in the epididymis in three of these high dose rats and in one additional high dose rat without testicular changes. The significance of this finding was uncertain. Systemic toxicity usually does not affect only one of a paired organ.
All other microscopic changes were considered to have occurred spontaneously and to be unrelated to test article administration. These changes generally occurred at single, low, or similar frequencies among the groups and their type, incidence or severity was not influenced by compound administration.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Litter size and weights: A decrease in the average number of pups per litter was observed at birth, and on day 4 post-partum in the high-dose group. Decreased litter weight and mean pup weight in the high-dose group (not statistically significant) were noted at birth and on day 4 post-partum.

- Number viable: Pup survival to day 4 was decreased in the high-dose group. The number of females with live pups on day 4 post-partum was 6, 8, 6, and 3 in the control, low-, mid- and high-dose groups, respectively. A statistically significant increase in pup mortality (females and total) from day 0 to day 4 was noted in the high dose group. Similar results were also seen in male pups but the difference was not statistically significant.

- Sex ratio: Sex ratios at birth and on day 4 post-partum did not show any differences between control and treated groups when calculated as the percentage of males.

- Abnormalities: Six pups in one litter of the high-dose group showed for/hindlimb digits missing or not defined during pre-weaning observations and/or necropsy. Necropsy examination of four of these pups confirmed the diagnoses of agenesis and/or microdactily. The other two pups were not available for necropsy. One pup from a different litter had flexure of the hindlimbs. These findings were considered treatment-related since they were rare findings in this species.
Alterations of bent tail and malrotated limbs were not considered to be treatment-related findings. Bent tail was noted at necropsy in three pups (1 litter) in the control group, in three pups (1 litter) of mid-dose, and 1 pup (1 litter) of the high dose groups on Day 4 post-partum. Malrotated hindlimbs were noted in 1 pup each in the control, low and mid-dose groups only. These alterations were observed in both the control and treated groups and there was no obvious dose response.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryo-fetal toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The relevance of the observed effects (decreased fertility indices and increased pre-birth losses,

embryo-fetal toxicity as well as developmental effects of microdactyly and/or agenesis of digits (one litter) and

flexure of hind limbs (one litter)) with regard to the hazard assessment

(i.e., classification) is unclear due to the following concerns about the study:

 Low fertility rate for the control group which limits the predictive (i.e., statistical)

power of the study as well as its reliability

 Lack of a clear dose-response relationship (effects only in the high dose group);

Observed parental toxicity raises the concern that 800 mg/kg for the high dose level

may have been too high

 Inadequate sample size for evaluation of fetal malformations in the high dose

group

 Lack of the historical control data in the study report does not allow for

distinguishing between substance-related and spontaneous effects

 Some incorrect values stated in the final report call into question the QA process

for the study, the data collection software, and /or perhaps some of the results

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

OECD 414, rat, gavage

A study was conducted (RTC, 2013) according to OECD 414 guideline and GLP. The study investigated the effects of Bis-(2-(2-butoxyethoxy)-ethoxy)-methane on pregnancy and embryo-foetal development in the Sprague Dawley rat following oral administration, according to the following experimental design:

 

Group	Treatment	Number of
Number	(mg/kg/day)+	Animals
1 2 3 4	0 40 120 400	24 F 24 F 24 F 24 F
+: in terms of test item as supplied F: females

 

Females were dosed starting from Day 6 until Day 19 post coitum.

The test item was administered, by the oral route, at a dose volume of 10 mL/kg.

The vehicle was an aqueous solution of carboxymethylcellulose (CMC 0.5% w/v).

The following investigations were performed: mortality check, clinical signs, body weight, food consumption and macroscopic observation of the females.

On Day 20 post coitum at necropsy, gravid uterus, corrected body weight (terminal body weight minus gravid uterus weight), corrected body weight gain (terminal body weight minus body weight at Day 6 post coitum minus gravid uterus weight), corpora lutea, implantation sites, number, sex and weight of all live foetuses were determined. Gross evaluation of the placenta was also performed.

 

Mortality and fate of females

No mortality of females occurred during the study.

One control female and one mid-dose female were found not pregnant at necropsy.

The number of females with live foetuses on Day 20 post coitum was 23 in the control group, 24 in the low dose group (40 mg/kg/day), 23 in the mid-dose group (120 mg/kg/day) and 24 in the high dose group (400 mg/kg/day).

 

Clinical signs

Treatment-related clinical signs noted in females receiving 400 mg/kg bw/day were ataxia, salivation and lethargic aspect. Other clinical signs, such as tremors, bradypnoea and piloerection were also noted.

One female at 120 mg/kg bw/day showed prone position and salivation on one day.

Females receiving 40 mg/kg/day did not show any clinical signs.

 

Body weight and body weight gain of females

A statistically significant decrease in body weight gain (approximately 35%) was observed on Day 9 post coitum in treated females receiving 400 mg/kg bw/day.

 

Food consumption

A slight decrease on food consumption was noted in females receiving 400 mg/kg bw/day on Day 9 post coitum.

 

Terminal body weight, uterus weight, corrected body weight and corrected body weight gain of females

 

A decrease in body weight gain from Day 6 post coitum and Day 20 post coitum corrected for gravid uterus weight was recorded in treated females receiving 400 mg/kg bw/day (approximately 20%).

 

Litter data and sex ratios

Litter data, mean foetal weight and sex ratios were not affected by treatment.

 

Macroscopic observations of females

No macroscopic observations were observed in females sacrificed on Day 20 post coitum on control and treated groups.

 

External examination of foetuses

A total of 6 small foetuses (foetal weight < 2.7 g) were detected: 1 out of 341 in the control group, 3 out of 357 in females receiving 40 mg/kg bw/day, 2 out of 345 in females receiving 120 mg/kg bw/day. One dead foetus was observed in a low dose litter (40 mg/kg bw/day) and a foetus showing cranioschisis was noted in another low dose litter. This malformation was considered incidental.

 

Fixed visceral examination of foetuses

No findings that could be considered treatment-related were noted at visceral examination of the foetuses in any group.

 

Skeletal examination of foetuses

Most of the alterations recorded at skeletal examinations were noted both in control and treated groups, with a similar incidence.

The foetus in the low dose group showing cranioschisis (see above) showed multiple malformations at skeletal examination. Unossified pubis was observed in another low dose foetus of a different litter.Considering that these alterations were observed in small foetuses (foetal weight<2.7 g), without dose-relation, they were considered incidental and not related to treatment.

 

Conclusions

On the basis of the results obtained in this study, Bis-(2-(2-butoxyethoxy)-ethoxy)-methane induced maternal toxicity at the dose level of 400 mg/kg bw/day, as shown by clinical signs (e.g., ataxia, tremors, etc.), and decreased body weight gain. Clinical signs at
120 mg/kg bw/day occurred in one animal only and were confined to salivation (Day 8 post coitum) and prone position (Day 10 post coitum) noted on one day. Due to the isolated and transient occurrence, these findings were not considered to be toxicologically relevant.

 

Therefore, the mid-dose level (120 mg/kg bw/day) was considered the NOAEL (No Observed Adverse Effect Level) for maternal toxicity.

The NOAEL for developmental toxicity and teratogenicity was considered to be 400 mg/kg bw/day.

 

 

OECD 414, rabbit, gavage

The effects of Bis(2-(2-butoxyethoxy)ethoxy)methane were further 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.

Justification for classification or non-classification

Based on the available data on the reproductive toxicity of Bis-(2-(2-butoxyethoxy)-ethoxy)-methane no classification is warranted according to the criteria of both, 67/548/EEC and CLP regulation (EC) 1272/2008.