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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Pivalic acid
EC Number:
200-922-5
EC Name:
Pivalic acid
Cas Number:
75-98-9
Molecular formula:
C5H10O2
IUPAC Name:
pivalic acid
Details on test material:
- Name of test material (as cited in study report): MRD-64-1
- Substance type: white crystalline powder
Specific details on test material used for the study:
Test item: Pivalic Acid
Test item identity (including alternative names): 2,2-dimethylpropanoic acid, VERSATIC™ Acid 5.
CAS number: 75-98-9
Intended use: Industrial Chemical, used in many chemical manufacturing processes.
Appearance: White crystalline solid at 20℃.
Storage conditions: At ambient temperature (15 to 25℃).
Supplier: Sponsor
Batch number: PV8A0174
Expiry date: Technical data sheet states that the shelf life should be three years starting from the manufactured date.
Manufactured date: 30 January 2018.
Physical state Solid at 20℃, liquid at 35℃.
Purity: 99.4%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Strain/Species: Sprague Dawley Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals ordered: 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation): 76 to 89 days old.
Weight range of the animals at the start of the study (Day 0 of gestation): 222 to 293 g.

Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24C and 40-70%. There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages constituting each group were blocked by group and mounted in batteries.
Bedding: Solid bottom cages contained softwood-based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage: Acclimatization up to four animals, During pairing one (stock) male and one female, Gestation one female

Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted.
3.4.5 Water Supply
Supply Potable water from the public supply via polycarbonate
bottles with sipper tubes. Bottles were changed at
appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% methylcellulose with 0.1% Tween 80
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity
The homogeneity and stability of formulations during storage were confirmed as part of another study, Covance Study number MN29GC. In that study, formulations in the range 1 to 200 mg/mL were determined to be stable for:
 15 days at ambient temperature (15 to 25°C).
 15 days when stored refrigerated (2 to 8°C).

Achieved concentration
Samples of each of the first and last formulation preparations were analyzed for achieved concentration of the test item.

Analysis
The method of analysis and results are presented in Attachment 13.2.
Details on mating procedure:
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating
Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after
mating, once daily at approximately the same time each day.
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
Frequency: Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
The pH of the formulations was low (acidic). Therefore, following filling of the dose equipment for each animal, the catheter was wiped dry, dipped in tap water, wiped dry and dipped in tap water again, before administration, to prevent any unwanted animal response during the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Examinations

Maternal examinations:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases as described in Section 3.7.

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus: Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn Number of:
Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Apparently non-pregnant animals and for apparently empty uterine horns

The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, 1964)).
Fetal examinations:
Examination of all viable fetuses and placentae

Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter

Sexed internally and eviscerated

Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.
Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.
Statistics:
For 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.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights
Ano-genital distance, average for each litter adjusted for litter average fetal body weight
Organ weights, absolute and adjusted for terminal body weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4

The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights and organ weight data:
Indices:
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) x 100 /Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations - Number of live fetuses) x 100 /Number of implantations

All group values and SD were calculated from the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Description (incidence and severity):
For surviving females receiving 225 mg/kg/day, treatment was associated with mean body weight loss during the first three days of treatment. Thereafter, mean body weight gains tended to be lower than control throughout gestation, although differences during late gestation were influenced, to some extent, by a lower contribution from the gravid uterus, due to lower mean litter size at this dose level. Lower body weight, compared with the control, achieved statistical significance on Day 13 of gestation and generally remained statistically significantly lower than control to termination. Overall body weight from the commencement of treatment, including when adjusted for the contribution of the gravid uterus, was statistically significantly lower than control.
For females receiving 75 mg/kg/day, treatment was associated with mean body weight loss during the first day of treatment; thereafter, mean body weight gains were generally similar to control. Overall body weight from the commencement of treatment was similar to control, however, when adjusted for the contribution of the gravid uterus, overall body weight gain was statistically significantly lower than control, suggesting a slight underlying effect on maternal body weight.
For females receiving 25 mg/kg/day, body weight gain was low, when compared with the control during the first day of treatment; thereafter, mean body weight gains were generally similar to control. Overall body weight from the commencement of treatment, including when adjusted for the contribution of the gravid uterus, was similar to control.
Description (incidence):
Three premature death occurred during this study.
One control female (Animal No. 8) was killed for animal welfare considerations on Day 19 of gestation due to general poor clinical condition. Prior to dispatch, this animal displayed labored breathing, piloerection, whole body pallor and had a red discharge from the vaginal area. At macroscopic examination, the female was found to be pregnant with 16 live young and all examined tissues were within normal limits.
Animal No. 63 that received 225 mg/kg/day showed abnormal breathing (gasping) and was cold to touch on Day 10 of gestation (after four consecutive days of treatment) and was killed for animal welfare consideration in the animal unit prior to dispatch to necropsy. At macroscopic examination this animal had a perforated esophagus, one dark area on the serous membrane of the esophagus and was pregnant with 16 live young. The cause of death was not believed to be treatment related but due to an error during the dose administration procedure.
Animal No. 79 that received 225 mg/kg bw/day was killed for animal welfare consideration on Day 17 of gestation (after 12 consecutive days of treatment) due to marked breathing impairment, characterised by labored and gasping respiration. This animal had shown dry rales from Day 15 and wet rales from Day 16, with chin rubbing and piloerection also being observed on Day 17. Body weight loss had been apparent for this animal from Day 15 of gestation. Prior to this, the female had shown decreased activity, labored and gasping respiration, piloerection and wet rales on Day 8, with piloerection continuing to Day 9 and wet rales persisting to Day 1l. Additionally, this animal was noted to be irritable during Days 9 to 11 and vocalisation was recorded during Days 9 to 12 and 15 to 17. At macroscopic examination, the female showed total litter resorption (15 early resorptions in uterus), but there were no other macroscopic findings.
Description (incidence and severity):
low, compared with controls, during the first four days of treatment (Days 6 to 10) and, despite food intake showing some improvement, remained statistically significantly low during the following four days of treatment (Days 10 to 14). Subsequent food intake for Days 14 to 18 was then similar to control. Although food consumption during Days 18 to 20 was statistically significantly lower than control, food intake at this stage of the gestation may have been influenced by lower mean litter size at this dose level.
Females receiving 75 mg/kg/day had significantly low food consumption immediately after the start of treatment for four days and this attained statistical difference from the controls. From Day 10 onwards, food consumption for these females was similar to control females. Mean food consumption for females receiving 25 mg/kg/day was similar to control females throughout the treatment period.
Description (incidence and severity):
The group mean absolute and terminal body weight adjusted thyroid (with parathyroids) weights of females given 25, 75 or 225 mg/kg/day were similar to control females.
Description (incidence and severity):
Findings observed at macroscopic necropsy examination were restricted to small thyroids for two control females and one female treated at 75 mg/kg/day. These isolated findings, in the absence of any effects on thyroid weights or histopathology were clearly incidental and of no biological significance.
Description (incidence and severity):
No microscopic findings were noted in the thyroids of females surviving to terminal necropsy.
Microscopic findings for examined tissues on this study were restricted to minimal pelvic dilatation of one kidney for a female treated at 75 mg/kg/day. This finding is commonly observed for rats of this strain and its isolated occurrence in this single animal was considered incidental and of no toxicological significance.

Maternal developmental toxicity

Description (incidence and severity):
One female (Animal No. 77) given 225 mg/kg/day was found to be not pregnant at necropsy and had complete litter resorption. Total litter resorption was also apparent in a decedent female (Animal No. 79) at this dose level.
Mean post-implantation loss, compared to control, was statistically significantly increased for females with live litter on Day 20 of gestation at 225 mg/kg/day, as a result of higher mean numbers of early and later resorption, leading to a lower live litter size at this dose level. This adverse effect on in-utero survival has to be viewed in the light of two other females at this dose level that showed total litter resorption. Mean sex ratio was similar to control and did not indicate any selective effect on survival for either sex. Mean numbers of corpora lutea and implantations and mean pre-implantation loss for these females (established prior to treatment) were similar to that of the control indicating that there was no pre-existing reproductive deficiency for animals at this dose level.
On Day 20 of gestation, mean numbers of corpora lutea, implantations, resorptions, implantation losses, the number of live young and sex ratio were similar for control females and females that received 25 or 75 mg/kg/day and there was no indication that maternal treatment at 25 or75 mg/kg/day had affected in utero offspring survival.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
mortality

Results (fetuses)

Description (incidence and severity):
At 225 mg/kg/day, mean fetal weights (male, female and combined) were low, when compared with control, with differences attaining statistical significance. The lower fetal weights, in combination with the lower litter size, at this dose level, resulted in lower mean litter weight, although this did not attain statistical significance. Mean placental weight was only slightly lower than control and appeared unaffected by maternal treatment.
Placental, total litter and fetal weights were unaffected by maternal treatment at 25 or 75 mg/kg/day
Details on embryotoxic / teratogenic effects:
At 225 mg/kg/day there was an increase in incidence of major abnormalities, in particular partially split sternum, heart and major vessel abnormalities and bent long bones/scapula(e) compared to concurrent control and, although at low incidence, still outside of HCD range.
Bent long bones and scapula(e) however can be related to low bodyweight and are thought to be transient in nature (Kimmel et al). All fetuses with the mentioned major abnormalities were of low bodyweight (between 2.46g and 2.72g)
At 225 mg/kg/day there was an increase in incidence of enlarged isthmus/absent lobe of thyroid (outside of HCD), partially undescended lobe of thymus and shiny skin (within HCD) all of which are related to low bodyweight and fetal immaturity, a transient stage in fetal development and not considered adverse.
Across the treated groups there was an increase in incidence of delayed ossification affecting the cranial centres (including the nasofrontal suture), sternebrae, cervical/thoracic/sacrocaudal vertebrae, metacarpals/metatarsals and pelvic bones which are all outside of HCD range.
Incomplete ossification is also a transient stage in fetal development, indicative of fetal immaturity and may be associated with a decrease in mean fetal weight. A significant decrease in mean fetal weight was observed at 225 mg/kg/day only.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights
changes in postnatal survival

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
225 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
Within this study, treatment at 225 mg/kg/day was associated with effects on body weight gain and food consumption and there was one death, the etiology of which was unclear. Collectively these findings were considered to preclude this dose level as representing a No Observed Adverse Effect Level (NOAEL). Effects observed for body weight and food consumption of females receiving 75 mg/kg/day were not considered adverse, therefore this dose level was considered to represent a NOAEL for the pregnant female.

At 225 mg/kg/day, maternal treatment was associated with adverse effects on embryofetal survival and foetal weights and, whilst findings from detailed examination of the foetuses mainly reflected foetal immaturity and were transient in nature, this dose level was considered not to represent a NOAEL for the developing conceptus. There were no effects of maternal treatment at 75 mg/kg/day on post-implantation survival or foetal weights and findings observed at detailed examination of the foetuses were considered to be transient in nature and not adverse. The NOAEL for embryo survival, growth and development was, therefore, considered to be 75 mg/kg/day.
Executive summary:

The purpose of this study was to assess the influence of Pivalic acid (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley rat according to TG OECD 414.

Three groups of 20 females received Pivalic acid at doses of 25, 75 or 225 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose with 0.1% Tween 80 at the same volume dose as treated groups, over the same treatment period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating including assessment of the uterine contents, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigation of the thyroid was also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

Three premature death occurred during this study. One control female was killed for welfare reasons on Day 19 of gestation due to general poor clinical condition. Two females given 225 mg/kg/day were killed for animal welfare considerations on Days 10 or 17 of gestation. The female killed on Day 10 had a perforated esophagus and a dark area on the serous membrane of the esophagus and was pregnant with 16 live young. This death was not treatment related but most likely caused by dosing trauma. The female killed on Day 17 of gestation had marked breathing impairment (labored breathing and gasping). This female was observed previously with wet/dry rales, chin rubbing and piloerection and had lost weight in the two days prior to dispatch. At macroscopic examination, the female was not pregnant (total litter resorption) and there were no macroscopic findings.  

There were no signs in relation to dosing or at routine physical examination in females receiving 25 or 75 mg/kg/day, but signs were seen in females receiving 225 mg/kg/day consisting of wet/dry rales and piloerection.  

The results of the thyroid hormone investigations showed that mean serum T3 concentrations were slightly low in females receiving 275 mg/kg/day compared with controls. TSH and T4 serum concentrations were unaffected by treatment.  

Body weight gain and food consumption were low in females receiving 75 or 225 mg/kg/day at the start of treatment, but improvement was then seen in females receiving 75 mg/kg/day but not in females receiving 225 mg/kg/day. Food consumption and body weight gain were unaffected by treatment in females receiving 25 mg/kg/day.  

There was no effect of treatment with Pivalic acid on thyroid and parathyroid weights, no macroscopic findings and no histopathological findings in the thyroids attributed to treatment at any dose level.  

There was no adverse effect of maternal treatment on the mean numbers of corpora lutea, implantations, resorptions, live young, the extent of pre- and post-implantation losses, sex ratio, ano-genital distance or on fetal and litter weights, and there were no major or minor abnormalities or skeletal variants considered related to treatment in females receiving 25 or 75 mg/kg/day.  

There was an adverse effect on in-utero survival in females receiving 225 mg/kg/day with higher than control post-implantation losses as a result of higher early and late resorptions, which led to lower live litter size at this dose level. In addition, the mean fetal weights (male, female and combined) were low, when compared with control, with differences attaining statistical significance. The mean placental weight, mean sex ratio and ano-genital distance was unaffected by treatment and the mean numbers of corpora lutea and implantations and pre-implantation loss for these females was similar to that of controls.  

At 225 mg/kg/day there was an increase in incidence of major abnormalities, in particular partially split sternum, heart and major vessel abnormalities and bent long bones/scapula(e)compared to concurrent control and, although at low incidence, these were still outside of the historical control (HCD) range. Bent long bones and scapula(e) however can be related to low bodyweight and are thought to be transient in nature (Kimmel et al). All fetuses with the mentioned major abnormalities were of low bodyweight (between 2.46g and 2.72g).

At 225 mg/kg/day there was an increase in incidence of enlarged isthmus/absent lobe of thyroid (outside of HCD), partially undescended lobe of thymus and shiny skin (within HCD) all of which are related to low bodyweight and fetal immaturity, a transient stage in fetal development and not considered adverse.

Across the treated groups there was an increase in incidence of delayed ossification affecting the cranial centres (including the nasofrontal suture), sternebrae, cervical/thoracic/sacrocaudal vertebrae, metacarpals/metatarsals and pelvic bones which were all outside of the HCD range.  

Incomplete ossification is also a transient stage in fetal development, indicative of fetal immaturity and may be associated with a decrease in mean fetal weight. A significant decrease in mean fetal weight was observed at 225 mg/kg/day only.

Conclusion

Within this study, treatment at 225 mg/kg/day was associated with effects on body weight gain and food consumption and there was one death, the etiology of which was unclear. Collectively these findings were considered to preclude this dose level as representing a No Observed Adverse Effect Level (NOAEL). Effects observed for body weight and food consumption of females receiving 75 mg/kg/day were not considered adverse, therefore this dose level was considered to represent a NOAEL for the pregnant female.

At 225 mg/kg/day, maternal treatment was associated with adverse effects on embryofetal survival and foetal weights and, whilst findings from detailed examination of the foetuses mainly reflected foetal immaturity and were transient in nature, this dose level was considered not to represent a NOAEL for the developing conceptus. There were no effects of maternal treatment at 75 mg/kg/day on post-implantation survival or foetal weights and findings observed at detailed examination of the foetuses were considered to be transient in nature and not adverse. The NOAEL for embryo survival, growth and development was, therefore, considered to be 75 mg/kg/day.

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