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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 September 2018 to 25 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-[methylenebis(oxy)]dibutane
EC Number:
219-909-0
EC Name:
1,1'-[methylenebis(oxy)]dibutane
Cas Number:
2568-90-3
Molecular formula:
C9H20O2
IUPAC Name:
1-(butoxymethoxy)butane

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 69 to 76 days old; females: 83 to 89 days old
- Weight at study initiation: (P) Males: 283-373 g; Females: 251-304 g
- Fasting period before study: no
- Housing: Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter
- Diet: ad libitum SDS VRF1 Certified pelleted diet
- Water: ad libitum Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Males: six days prior to the commencement of treatment. Females: 20 days prior to the commencement of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark
IN-LIFE DATES: From: 10 October 2018 To: 22 December 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Starting with the lowest concentration, the required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5ml/kg
Details on mating procedure:
- M/F ratio per cage: 1:1 from within the same treatment groups
- Length of cohabitation: Up to two weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear. Day 0 of gestation: When positive evidence of mating was detected
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.
The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 100 μg/mL.
For the First and Last Week of dosing, all groups were sampled (4 × 1 mL, accurately weighed) from the middle of the formulation by Pharmacy personnel.
Two samples from each group were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once results were obtained.
Procedural recoveries were prepared as a quality control measure and were not used to correct for the formulation samples.
The mean concentrations of Butylal in test formulations analyzed during the study and the deviation of the mean result from the nominal value are detailed in Table 1.
The mean concentrations were within +10/-15% of the nominal concentration, confirming accurate formulation. The percentage difference from mean values remained within 2%, confirming precise analysis.
Procedural recovery values were within the validated range confirming the continued accuracy of the analytical procedure.
Duration of treatment / exposure:
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Males: Two weeks pre-pairing up to necropsy after minimum of four weeks
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation
Frequency of treatment:
Once daily at approximately the same time each day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 0, 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on findings from a preliminary 4-week repeat dose toxicity study in the Sprague-Dawley rat.
In the 4-week preliminary study dose levels of 500, 750, and 1000 mg/kg/day had no effect on general condition, body weight, food consumption or macropathology. Effects were limited to high liver weights for males that received 1000 mg/kg/day and for females at all dose levels.
It was therefore considered that a high dose of 1000 mg/kg/day would be tolerated in this OECD 421 screening study. The low and intermediate doses were 100 and 300 mg/kg/day, providing approximate 3-fold dose increments and allowing investigation of any dose-related response.
- Rationale for animal assignment: On arrival and non-selective allocation to cages

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation, One to two hours after completion of dosing, As late as possible in the working day. A detailed physical examination was performed on each animal to monitor general health according to the following schedule: F0 males weekly; F0 females: Week 1 and 2 - weekly, Gestation phase - Days 0, 7, 14 and 20, Lactation phase - Days 1, 7 and 13

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of animals was recorded as follows
F0 males: Weekly during acclimatization (not reported), Before dosing on the day that treatment commenced (Day 1) and weekly thereafter; On the day of necropsy.
F0 females: Weekly during acclimatization (not reported), Before dosing on the day that treatment commenced (Day 1) and weekly before pairing, Days 0, 7, 14 and 20 after mating, Day 1, 7 and 13 of lactation, On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly before pairing.
For females after mating food consumption was recorded as follows:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

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

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
- After pairing until mating.
- For four days before scheduled termination (nominally Days 10 to 13 of lactation).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, thyroid hormone analysis. Particular attention was paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content
Postmortem examinations (parental animals):
SACRIFICE
Sequence: To allow satisfactory inter-group comparison

GROSS NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected for thyroid hormone analysis: Scheduled kill - Day 13 of age
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
F1 offspring on Day 4 of age: Externally normal offspring were discarded without examination. Externally abnormal offspring were subject to an external macroscopic examination and retained pending possible future examination.
F1 offspring on Day 13 of age: All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative. Thyroid glands were preserved from two offspring per litter, one male and one female in each litter, where possible.
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre coital interval, mating performance, gestation length and index and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.
Reproductive indices:
Estrous Cycle
The incidence and percentage females showing the following classifications of estrous cycles before treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination.
Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.
Mating Performance and Fertility
Individual data were tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating x 100) / Animals paired

Conception rate (%) = (Number of animals achieving pregnancy x 100) / Animals mated
Fertility index (%) = (Number of animals achieving pregnancy x 100) /Animals paired

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (Number of live litters born x 100) / Number pregnant
Offspring viability indices:
The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born x 100) / Total number of uterine implantation sites

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering x 100) / Total number of offspring born

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) x 100) / Number live offspring on Day 1 after littering

Lactation index (%) = (Number of live offspring on Day 13 after littering x 100) / Number of live offspring on Day 4 (after blood sampling)


Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs at routine physical examination that could be related to treatment.
Signs associated with dose administration were limited to a low incidence of increased salivation with associated chin rubbing for animals receiving 1000 mg/kg/day; these signs are often seen in association with dosing by oral gavage and are considered to relate to the palatability of the formulations rather than a direct effect of treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain for males during treatment and for females before pairing was unaffected by treatment.
During early gestation (GD0-7) females receiving 1000 mg/kg/day showed significantly low weight gain when compared with Controls (p<0.05); however subsequent weight gain and the overall weight gain during gestation was essentially similar to Controls.
During lactation the absolute body weights for females receiving 1000 mg/kg/day on LD1 and LD7 were slightly but significantly low when compared with Controls (p<0.05). Body weight gain (LD7-13 and LD1-13) for females at 1000 mg/kg/day was high when compared with Controls although the difference did not attain statistical significance.
Body weight gain at 100 and 300 mg/kg/day was unaffected by treatment during both gestation and lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for the two-week period before pairing was unaffected by treatment.
During GD14-19 food consumption for females receiving 1000 mg/kg/day was low when compared with Controls with the difference attaining statistical significance (p<0.01).
During lactation food consumption for females receiving 1000 mg/kg/day was slightly low at approximately 94% of Controls; however, the differences for each recording period did not attain statistical significance.
Food consumption at 100 and 300 mg/kg/day was unaffected by treatment during both gestation and lactation.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Individual serum TSH concentrations were found to be variable.
Both male and female mean serum TSH concentrations were slightly low in the groups that received Butylal via oral gavage when compared with the Control Group. However, these differences did not attain statistical significance.
Thyroxine levels in females that received 1000 mg/kg/day and in F1 offspring on Day 13 of age at 300 or 1000 mg/kg/day were slightly low when compared with Controls; this was not evident in F0 males or F1 female offspring on Day 4 of age. The data was highly variable and the differences from Control were slight, therefore there was no conclusive evidence that could attribute this to treatment with Butylal.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The microscopic examination performed after at least 4 weeks of treatment revealed no test item related lesions in the tissues examined (testes, epididymides and ovaries).
Incidental Findings:
All histological changes were considered to be unrelated to treatment.
One Group 3 female had a mammary adenocarcinoma which is an incidental finding and not unusual in female rats of this age.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment. At termination all females were confirmed to be in diestrus.
Reproductive performance:
no effects observed
Description (incidence and severity):
Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment. At termination all females were confirmed to be in diestrus.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs that were considered to relate to treatment
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of implantations for females receiving 1000 mg/kg/day was slightly but significantly high when compared with Controls; subsequent litter size was slightly high but these differences did not attain statistical significance. This difference was not considered to be of any toxicological significance.
Litter size at 100 or 300 mg/kg/day and offspring survival at all dose levels was unaffected by parental treatment.
The mean percentage of male offspring in the treated groups was slightly but significantly low when compared with Controls on Day 1 and 4 of age (the values on Day 4 and 13 after females are selected to provide blood samples of T4 analysis are not considered relevant); however this is considered to be unrelated to treatment as the Control mean was atypically high.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day the absolute mean body weight for offspring on Day 1 of age (p<0.01) and subsequent mean weight gain up to termination on Day 13 of age was low when compared with Controls (80% of Controls for male offspring, p<0.05, and 84% of Controls for female offspring); the mean values at 1000 mg/kg/day exceeded the historical control range.
Offspring body weight at 100 or 300 mg/kg/day was unaffected by parental treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance for both male and female offspring was unaffected by parental treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were apparent for male offspring on Day 13 of age
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that either died prematurely or at scheduled termination did not reveal any findings that could be related to parental treatment

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Treatment of Butylal to parental animals at 100, 300 or 1000 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, thyroid hormones, estrus cycles, mating performance, fertility, macropathology or histopathology. The clinical condition and survival of the subsequent F1 offspring were also unaffected by parental treatment.
Adverse findings were limited to offspring derived from the group that received 1000 mg/kg/day which had low absolute weight and bodyweight gain from Day 1 of age. This effect on offspring bodyweight was in the absence of any effect on offspring survival, general condition or thyroid hormones and within the context of this screening study is considered to not warrant the classification of the test item as a reproductive toxicant.

It was therefore concluded that the no observed adverse effect level (NOAEL) was 1000 mg/kg/day for parental systemic toxicity and reproductive parental toxicity. For offspring development 1000 mg/kg/day was considered to be the lowest observed adverse effect level (LOAEL) with a NOAEL of 300 mg/kg/day.
Executive summary:

The purpose of this study was a screening test for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, Butylal, by oral gavage administration for at least four weeks.


Three groups of ten male and ten female rats received Butylal at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration.  Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks.  Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation.  Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation.  The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk.  A similarly constituted Control group received the vehicle, corn oil, at the same volume dose (5 mL/kg/day) as the treated groups.


During the study, clinical condition, body weight, food consumption, estrous cycles, pre‑coital interval, mating performance, fertility, gestation length, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.


The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed.  Nipple counts were performed on male offspring on Day 13 of age.  Blood samples were collected from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis. 


Results


Parental responses


General condition, estrous cycles, mating performance, fertility, gestation length/index, thyroid hormones,  macropathology, organ weights and histopathology were unaffected by treatment at dose levels up to and including 1000 mg/kg/day.


Body weight and food consumption during the two week period prior to pairing for mating was unaffected by treatment at dose levels up to and including 1000 mg/kg/day.


During gestation females receiving 1000 mg/kg/day showed low weight gain during the first week of gestation (GD0-7); however overall body weight gain during gestation was similar to Controls.


During lactation females receiving 1000 mg/kg/day had low absolute body weight during Week 1 (LD1 and LD7); these females showed slightly high weight gain during Week 2 of lactation, resulting in a mean body weight on Day 13 that was similar to Controls.


At 1000 mg/kg/day females showed low food consumption during the last week of gestation (GD14-19) and the last week of lactation (LD7-13).


Bodyweight and food consumption after mating and during lactation for females receiving 100 or 300 mg/kg/day was unaffected by treatment.


Litter responses


Litter size, offspring survival, sex ratio, ano-genital distance, general condition, thyroid hormones and macropathology showed no adverse effects of parental treatment.


At 1000 mg/kg/day the absolute mean body weight for offspring on Day 1 of age  and subsequent mean weight gain up to termination on Day 13 of age was low when compared with Controls (80% of Controls for male offspring, p<0.05, and 84% of Controls for female offspring)


Offspring body weight at 100 or 300 mg/kg/day was unaffected by parental treatment.


Conclusion


Treatment of Butylal to parental animals at 100, 300 or 1000 mg/kg/day for two weeks before pairing, during pairing  and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was well tolerated.  There was no adverse effect on parental clinical condition, body weight performance, food consumption, thyroid hormones, estrus cycles, mating performance, fertility, macropathology or histopathology.  The clinical condition and survival of the subsequent F1 offspring were also unaffected by parental treatment.


Adverse findings were limited to offspring derived from the group that received 1000 mg/kg/day which had low absolute weight and bodyweight gain from Day 1 of age. This effect on offspring bodyweight was in the absence of any effect on offspring survival, general condition or thyroid hormones and within the context of this screening study is considered to not warrant the classification of the test item as a reproductive toxicant.


 


It was therefore concluded that the no observed adverse effect level (NOAEL) was 1000 mg/kg/day for parental systemic toxicity and  reproductive parental toxicity.   For offspring development 1000 mg/kg/day was considered to be the lowest observed adverse effect level (LOAEL) with a NOAEL of 300 mg/kg/day.

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