Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results of 90-day study and three developmental studies in rats, no adverse effects on reproductive function and reproductive performance can be attributed to the test material. NOAEL of 500 mg/kg bw established in the 90-day study in rats is considered appropriate for reproductive toxicity of NBP.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Endpoint:
toxicity to reproduction
Remarks:
other: 90-day study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-01-16 to 2014-10-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 408
Deviations:
no
Principles of method if other than guideline:
Oestrous cycle assessment and sperm analysis.
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Han™: RccHan™: WIST
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: six to eight weeks old.
- Weight at study initiation: 187 to 241 g (males); 166 to 199 g (females)
- Housing: in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (in a single air-conditioned room).
- Diet (e.g. ad libitum): ad libitum (pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.))
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2014-01-30 (first day of treatment) To: 2014-05-01 (final day of necropsy).
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test item formulations were determined by the test facility. Results from the previous study show the formulations to be stable for at least nineteen days. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 2, 20 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL
Details on mating procedure:
Not applicable (this is 90-day study).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken and analysed for concentration of the test substance at the test facility. The results indicate that the prepared formulations were within ± 4% of the nominal concentration.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Details on study schedule:
Not applicable (this is 90-day study)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen in collaboration with the sponsor based on the results of previous toxicity work (Fourteen Day Range-Finding Toxicity Study, Harlan Laboratories Study Number: 41303992). Initial investigations at dose levels of 50, 200 and 500 mg/kg bw/day did not indicate any toxicologically significant effects of treatment; therefore subsequent sighting work at 750 and 1000 mg/kg bw/day was conducted.
At 1000 mg/kg bw/day animals were terminated after two days of dosing based on body weight loss in animals of either sex and clinical observations of ataxia, tiptoe gait, decreased respiratory rate, increased salivation and high stepping gait in the female.
At 750 mg/kg bw/day, animals were terminated following three days of dosing due to body weight loss apparent in the female, accompanied by clinical observations of ataxia, noisy respiration, laboured respiration, decreased respiratory rate and hunched posture. Consequently, these dose levels were considered to be unsuitable for further investigation in repeated dose toxicity studies.

- Rationale for animal assignment:
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
None.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of treatment and at weekly intervals thereafter. Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia,Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defaecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION
Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OTHER:
- Haematology, clinical chemistry and neurobehavioural examination (sensory activity / grip strength / motor activity; see section 7.5.1.)
Oestrous cyclicity (parental animals):
A vaginal smear was taken daily from females for 21 Days during the final 3 weeks of the study. The sample was placed on a glass slide, the smears were allowed to dry and were then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrus was recorded.
Sperm parameters (parental animals):
Parameters examined in all animals: [testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other: numbers of homogenisation resistant spermatids]
In details, at necropsy the following evaluations were performed on all males:
i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant spermatids.
iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. A minimum of 200 individual sperm were assessed using an automated semen analyser, to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
iv) A sample of semen was preserved in formalin and then stained with eosin. A sub-sample was placed on a glass slide with a coverslip and a morphometric analysis of sampled semen was performed manually.
v) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with a DNA specific fluorescent stain and a subsample was analysed for numbers of homogenisation resistant spermatids.

For ii), iv) and v) above, samples from Groups 1 and 4 were evaluated only as no significant effects were seen.
Litter observations:
Not applicable (this is 90-day study)
Postmortem examinations (parental animals):
SACRIFICE
- all animals: Day 91 (week 13)
GROSS PATHOLOGY: Yes
The following organs were weighed: adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, uterus, liver.
HISTOPATHOLOGY: Yes (see table 2)
All tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). All tissues from control and 500 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Since there were indications of treatment-related liver and thymus changes in high dose males and females and kidney and adrenal changes in the males, examination was subsequently extended to include similarly prepared sections of liver and thymus from low and intermediate dose group males and females and similarly prepared sections of kidney and adrenals from low and intermediate dose group males.
Postmortem examinations (offspring):
Not applicable (this is 90-day study)
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANOVA with appropriate covarities. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann- Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Reproductive indices:
Not applicable (this is 90-day study)
Offspring viability indices:
Not applicable (this is 90-day study)
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
macroscopic abnormalities were detected in liver, kidney, thymus and adrenals (adaptive responses without toxicological importance).
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
Description (incidence and severity):
not applicable (90-day study)
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths. Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 10, 100 or 500 mg/kg bw/day.
Animals of either sex treated with 500 mg/kg bw/day had an increased salivation post-dose from Day 21 (Females) and Day 23 (Males). Three males and six females treated with 100 mg/kg bw/day also showed isolated incidences of increased salivation. Four males and two females treated with 500 mg/kg bw/day also had isolated episodes of noisy respiration during the study period. Observations of this nature are commonly experienced following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.
One female treated with 500 mg/kg bw/day had chromodacryorrhea on Day 63 which in isolation was considered not to represent a toxicologically significant effect of treatment. This animal also had generalized fur loss between Days 76 and 77 which is commonly observed in group housed animals and in isolation was considered not to represent a toxicological effect of treatment.

BODY WEIGHT AND WEIGHT GAIN
There was no adverse effect of treatment on body weight development at 10, 100 or 500 mg/kg bw/day. Intergroup differences in body weight gains for males at 100 and 500 mg/kg bw/day and females at 500 mg/kg bw/day were considered to represent normal biological variation rather than an adverse effect of treatment. Males treated with 500 mg/kg bw/day showed lower body weight gain during Week 8 of the study, with differences from control achieving statistical significance. Recovery was evident during Week 9 with mean body weight gains exceeding that of control animals. Statistically significantly lower body weight gains were apparent during Weeks 10 and 11; however, recovery was evident thereafter with body weight gains superior to controls evident during the final week of treatment, where differences from control attained statistical significance. Overall body weight gain was comparable to control.
Females treated with 500 mg/kg bw/day showed a statistically significantly lower body weight gain during Week 11 of the study, recovery was evident thereafter and overall body weight gain was comparable to control animals. Males treated with 100 mg/kg bw/day showed a statistically significant reduction in body weight gain during Week 8 of the study, recovery was evident thereafter.

FOOD CONSUMPTION
No obvious effect on food consumption was detected at 10, 100 or 500 mg/kg bw/day.

FOOD EFFICIENCY
Animals of either sex treated with 500 mg/kg bw/day, males treated with 100 mg/kg bw/day and females treated with 10 mg/kg bw/day showed a slight reduction in food efficiency during Week 11. Females treated with 100 mg/kg bw/day showed a slight reduction in food efficiency during Weeks 8, 9 and 13. The differences in food efficiency observed were consistent with the intergroup differences in body weight gain observed.

REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
No adverse effects were detected during the oestrous cycle assessments. All control and treated females showed evidence of oestrus (see table 1).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no toxicologically significant effects detected in sperm concentration, morphological assessments or in homogenisation-resistant spermatid counts (see tables 2, 3 and 4).
Males treated with 500 mg/kg bw/day had a statistically significant reduction in the percentage of sperm with normal morphology accompanied by a statistically significant increase in the number of sperm with abnormal morphology. Due to the nature of the abnormalities observed, the higher number of morphological abnormalities were considered to represent artifactual abnormalities associated with the slide smearing procedure and as such the reduction in percentage of normal sperm and the increase in the percentage of abnormal sperm were considered not to represent an adverse effect of treatment.

ORGAN WEIGHTS
Animals of either sex treated with 500 mg/kg bw/day and males treated with 100 mg/kg bw/day had a statistically significant increase in liver weights, both absolute and relative to terminal body weight. Males treated with 500 and 100 mg/kg bw/day also showed a statistically significant increase in absolute and relative kidney weight. No such effects were detected in females treated with 100 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.
Males treated with 500 mg/kg bw/day showed a statistically significant increase in spleen weight, both absolute and relative to terminal body weight, in the absence of any microscopic abnormalities in the spleen, this intergroup difference was considered not to be toxicologically significant. Males treated with 100 mg/kg bw/day showed a statistically significant increase in brain weight, both absolute and relative to terminal body weight. The majority of individual values were within normal background ranges for rats of the strain and age used and therefore, the intergroup difference was considered not to be toxicologically significant.
Females treated with 500 mg/kg bw/day showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight. The majority of the individual values were within normal ranges for rats of the strain and age used and no histological effects were present in the kidneys of high dose females therefore the intergroup difference was considered not to be of toxicological significance.

GROSS PATHOLOGY
At 500 mg/kg bw/day, eight males had an enlarged liver. No toxicologically significant macroscopic abnormalities were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 100 or 10 mg/kg bw/day.
One male had a fluid filled right kidney with increased pelvic space at necropsy, in isolation and in absence of any supporting histopathological findings, this finding was considered not to be toxicologically significant. One female treated with 500 mg/kg bw/day, two females treated with 100 mg/kg bw/day and two females treated with 10 mg/kg bw/day had reddened lungs at necropsy, in the absence of any microscopic changes to suggest an effect of treatment in the lungs, these findings were considered not to be of toxicological importance. One female treated with 10 mg/kg bw/day also had dark kidneys, in the absence of any similar findings at 500 mg/kg bw/day and any supporting histopathological correlates, this finding was considered to be incidental and of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following macroscopic abnormalities were detected:

Liver: hypertrophy or centrilobular hypertrophy was present in 7/10 males and all females in the high dose group (500 mg/kg bw/day) along with 3/10 males in the intermediate dose group (100 mg/kg bw/day).

Kidneys: There was an increase in incidence and severity of hyaline droplet accumulation, multifocal basophilic tubules (degenerating/regenerating) and the presence of proteinaceous casts in the tubules of males only receiving 100 mg/kg bw/day and above.

Thymus: Atrophy, increased at a minimal level was present in males and females receiving 500 mg/kg bw/day and in males receiving 100 mg/kg bw/day.

Adrenals: Vacuolation in the adrenal cortex (mainly the zona fasciculata) was present in males only at an increased incidence and severity at 100 mg/kg bw/day and above. One male treated with 10 mg/kg bw/day had vacuolation above the expected level, however the toxicological significance of this in only one male is equivocal.

OTHER FINDINGS (PARENTAL ANIMALS)
- Haematology, clinical chemistry and neurobehavioural examination (sensory activity / grip strength / motor activity; see section 7.5.1.)
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: treatment related effects, but not adverse.
Remarks on result:
other: Generation: all animals (migrated information)
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: treatment related, but not adverse effects were observed at 100 and 500 mg/kg bw.
Remarks on result:
other: Generation: all animals (migrated information)
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: treatment related, but not adverse effects were observed at 500 mg/kg bw.
Remarks on result:
other: Generation: all animals (migrated information)
Clinical signs:
not examined
Description (incidence and severity):
not applicable (90-day study)
Mortality / viability:
not examined
Description (incidence and severity):
not applicable (90-day study)
Body weight and weight changes:
not examined
Description (incidence and severity):
not applicable (90-day study)
Sexual maturation:
not examined
Description (incidence and severity):
not applicable (90-day study)
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
not applicable (90-day study)
Gross pathological findings:
not examined
Description (incidence and severity):
not applicable (90-day study)
Histopathological findings:
not examined
Description (incidence and severity):
not applicable (90-day study)
not applicable (90-day study)
Remarks on result:
other: not applicable (90-day study)
Reproductive effects observed:
not specified

Table 1. Oestrous Cycle Assessment.

Group

Number of

Females

Number of Females with

Regular Cycle

Control

10

10

10 mg/kg bw

10

10

100 mg/kg bw

10

10

500 mg/kg bw

10

10

Table 2. Group Mean Sperm Concentration and Motility.

Group

Number of animals

Concentration (M/mL,
mean ± sd)

Motility (%,
mean ± sd)

Progressive motility (%,
mean ± sd)

Control

10

174.6 ± 58.9

72 ± 23

8 ± 4

10 mg/kg bw

10

158.0 ± 74.1

70 ± 23

7 ± 3

100 mg/kg bw

10

201.8 ± 43.5

79 ± 5

8 ± 4

500 mg/kg bw

10

229.9 ± 27.1

82 ± 7

7 ± 2

Table 3. Group Mean Sperm Morphology (mean ± sd).

Group

Number normal**

Number abnormal

Normal (%)

Abnormal (%)

Misshapen (%)

Reverse head (%)

Head only (%)

No head (%)

Short tail (%)

Control*

199 ±1.1

0.9 ± 1.1

99.6 ± 0.6

0.5 ± 0.6

0.0 ± 0.0

0.0 ± 0.0

0.4 ± 0.5

0.0 ± 0.0

0.1 ± 0.2

500 mg/kg bw

197.5 ± 2.0

2.5 ± 2.0

98.8 ± 1.0#

1.3 ± 1.0#

0.1 ± 0.2

0.1 ± 0.2

0.7 ± 0.7

0.2 ± 0.3

0.4 ± 0.5

*Number of animals: 10;

** Number counted: 200 sperms

# significantly different from control (p<0.05)

Table 4. Group Mean Homogenisation Resistant Spermatid Counts.

Group

Number of animals

Testis
(million/gram, mean ± sd)

Cauda epididymis
(million /gram, mean ± sd)

Control*

10

42.1 ± 7.5

411.8 ± 198.4

500 mg/kg bw

10

38.3 ± 6.5

397.9 ± 136.2

 

Conclusions:
The oral administration of the test substance to rats by gavage for a period of ninety consecutive days at dose levels of 10, 100 and 500 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 500 mg/kg bw/day and males treated with 100 mg/kg bw/day.

The following effects have been considered to be treatment related but adaptive in nature and therefore not adverse:
1. The microscopic liver changes in animals of either sex treated with 500 mg/kg bw/day and males treated with 100 mg/kg bw/day, and the associated blood chemistry changes identified in animals of either sex treated with 500 mg/kg bw/day were likely to represent an adaptive response to treatment and therefore considered not to represent a serious risk to health.
2. The microscopic changes in the adrenals of males treated with 500 and 100 mg/kg bw/day and the microscopic thymus changes are likely to result from the adaptive changes apparent in the liver or a secondary stress related response. In terms of extrapolation to man, and risk assessment calculations, the effects relating to renal changes in the male rat are species and sex specific and therefore are not relevant.

For these reasons, 500 mg/kg bw/day may be regarded as a 'No Observed Adverse Effect Level' (NOAEL) for animals of either sex. The No Observed Effect Level (NOEL) was considered to be 10 mg/kg bw/day for males and 100 mg/kg bw/day for females.
Executive summary:

The study was designed to investigate the systemic toxicity of N-Butylpyrrolidone and is compatible with the OECD guideline 408. The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 and 500 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water). The dose levels were chosen based on the results of previous fourteen day range-finding study (Harlan Laboratories Study Number: 41303992). Observations at 750 and 1000 mg/kg bw/day proved to be sufficiently adverse to exclude these dose levels from subsequent investigation. No toxicologically significant effects of treatment were apparent at 500 mg/kg bw/day, therefore this dose level was considered most suitable for investigation in studies of a longer duration. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths. Neither the type, nor incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 10, 100 or 500 mg/kg bw/day. There were no toxicologically significant changes in the behavioural parameters measured, functional performance tests and in sensor reactivity assessments. There was no adverse effect of treatment on body weight development at 10, 100 or 500 mg/kg bw/day. No obvious effect on food consumption or food efficiency was detected at 10, 100 or 500 mg/kg bw/day. Daily visual inspection of water bottles did not reveal any overt differences in water consumption at 10, 100 or 500 mg/kg bw/day.

There were no treatment-related ocular effects.

No adverse effects were detected during the oestrous cycle assessments. All control and treated females showed evidence of oestrus.

There were no toxicologically significant effects of treatment on the haematology parameters measured. Regarding blood chemistry parameters, Males treated with 500 mg/kg bw/day showed a statistically significant increase in total protein, calcium, creatinine and bile acid and a statistically significant reduction in albumin/globulin ratio. Females treated with 500 mg/kg bw/day showed a statistically significant reduction in albumin/globulin ratio, chloride and alkaline phosphatase and a statistically significant increase in cholesterol, bilirubin and bile acid. Males treated with 100 mg/kg bw/day showed a statistically significant increase in chloride. No such effects were detected in females treated with 100 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.

Eight males treated with 500 mg/kg bw/day had enlarged livers at necropsy. No toxicologically significant macroscopic findings were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 100 or 10 mg/kg bw/day.

There were no toxicologically significant effects detected in sperm concentration, morphological assessments or in homogenisation-resistant spermatid counts.

Animals of either sex treated with 500 mg/kg bw/day and males treated with 100 mg/kg bw/day had a statistically significant increase in kidney and liver weights, both absolute and relative to terminal body weight. No such effects were evident in females treated with 100 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.

The following macroscopic abnormalities were detected:

Liver: hypertrophy or centrilobular hypertrophy was present in 7/10 males and all females in the high dose group (500 mg/kg bw/day) along with 3/10 males in the intermediate dose group (100 mg/kg bw/day).

Kidneys: There was an increase in incidence and severity of hyaline droplet accumulation, multifocal basophilic tubules (degenerating/regenerating) and the presence of proteinaceous casts in the tubules of males only receiving 100 mg/kg bw/day and above.

Thymus: Atrophy, increased at a minimal level was present in males and females receiving 500 mg/kg bw/day and in males receiving 100 mg/kg bw/day.

Adrenals: Vacuolation in the adrenal cortex (mainly the zona fasciculata) was present in males only at an increased incidence and severity at 100 mg/kg bw/day and above. One male treated with 10 mg/kg bw/day had vacuolation above the expected level, however the toxicological significance of this in only one male is equivocal.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.
Additional information

Reproductive toxicity potential of N-Butylpyrrolidone can be assessed using the results of three developmental toxicity studies (OECD 414) by oral, inhalation and dermal routes of exposure and by the results of the available 90 -day study in which oestrus cycle, sperm parameters, weight, gross pathology and histhopathology of reproductive organs were analysed.

Developmental toxicity studies

No adverse effects of test substance administration on reproductive performance and function were noted in three developmental toxicity studies in rats.

In an inhalation developmental toxicity study (OECD 414; Charles River Laboratories, 2016c; Report No. WIL- 387081), the substance was administered via whole-body inhalation exposure to 3 groups of 24 bred female Crl:CD(SD) rats for 6 hours per day from Gestation Days 6–19. The exposure concentrations were 0.3, 0.6 and 1.2 mg/L. Reproductive parameters were not affected by the test substance administration at all exposure levels tested. Fertility, intrauterine survival, postimplantation loss, live litter size, fetal sex ratios, mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant. The NOAEC of 0.6 mg/L was established for maternal toxicity based on clinical signs, decreased body weight and food consumption in the highest dose group.

In an oral developmental toxicity study (OECD 414; Charles River Laboratories, 2016a; Report No. WIL - 387076), the test substance was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 200, 300, 400, and 500 mg/kg/day. Reproductive function was not affected by the test substance administration. Intrauterine growth in the 200 mg/kg/day group and survival in the 200, 300, 400, and 500 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. The NOAEL of 400 mg/kg bw was established for maternal toxicity based on clinical signs, decreased body weight and body weight gains and on decreased food consumption in the highest dose group.

In a dermal developmental toxicity study (OECD 414; Charles River Laboratories, 2016b; Report No. WIL - 387079), the test substance was administered by dermal application to 3 groups of bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Animals were exposed to the test substance for approximately 23 hours each day. Exposure levels were 375, 500, and 750 mg/kg/day. Reproductive parameters were not affected by the test substance administration. Postimplantation loss, live litter size, fetal sex ratios, mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant. The NOAEL of 500 mg/kg bw was established for maternal toxicity based on body weight losses and lower mean body weight gains with corresponding reduced mean food consumption in the highest dose group.

In a supporting developmental toxicity study (OECD 414; LPT, 2013c; Report No. 29197), the test substance was administered to female CD® / Crl:CD (SD) rats at dose levels of 5, 50 or 500 mg/kg b.w./day, orally by gavage from the 6th to 19th day of pregnancy.

Even though the study itself is considered reliable and valid, the results are considered not to allow a meaningful interpretation, due to the following reasons:

1) The selection of dose levels (5, 50 and 500 mg/kg bw) is based upon differences of a factor of ten (log order); moreover the dose range-finder had shown only minimal changes at 500 mg/kg/day in both the fetus and the dams, which already suggested that the NOAEL would be around 500;

2) No background malformation rate typically known for the strain used;

3) The reasoning for considering reduced fetal body weights as not related to treatment is not sufficiently justified.

In conclusion, even though, the data itself is valid, the poor choice of doses made a meaningful interpretation impossible.

The results of the dose range-finding study (OECD 414; LPT, 2013d; Report No. 29177) are also considered not to allow a meaningful interpretation due to the same reasons.
In the main study, no test substance-related influence was noted on the reproductive parameters of dams. The NOAEL of 500 mg/kg bw was established for reproductive toxicity since no test item-related influence was noted for the number of corpora lutea, implantation sites, resorptions and live and dead foetuses. The percentages of pre- and post-implantation loss were also not influenced by the test item. The NOAEL of 50 mg/kg bw was established for general toxicity because of reduced body weight and body weight gains of the dams and reduced food comption and gravid uterine weights in the highest dose group.

90 - Day study

In the 90-day study, N-Butylpyrrolidone was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 and 500 mg/kg bw/day (OECD 408, Harlan Laboratories, 2014b; Report No. 41303953). A control group of ten males and ten females was dosed with vehicle alone (Distilled water). Clinical signs, functional observations, body weight change, dietary intake, water consumption, ophthalmoscopic examination, haematology and blood chemistry were monitored during the study. Oestrous cycle assessment and an assessment of semen parameters were conducted in animals of all dose groups. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths. Neither the type, nor incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 10, 100 or 500 mg/kg bw/day. There were no toxicologically significant changes in the behavioural parameters measured, functional performance tests and in sensor reactivity assessments. There was no adverse effect of treatment on body weight development, food consumption and food efficiency at all dose levels tested. There were no treatment-related ocular effects. There were no toxicologically significant effects of treatment on the haematology parameters measured. Treatment related effects were observed in blood chemistry parameters, gross pathology, organ weights and histopathology (for details see section 7.5.1 of the IUCLID file or section 5.6 of the CSR). The changes were considered not to represent an adverse effect of treatment but were adaptive and therefore were considered not to be of toxicological importance.

Regarding reproduction function; no adverse effects were detected during the oestrous cycle assessments. All control and treated females showed evidence of oestrus. There were no toxicologically significant effects detected in sperm concentration, morphological assessments or in homogenisation-resistant spermatid counts. There were no abnormalities detected in reproductive organs, their weights and histopathology.

For general toxicity, 500 mg/kg bw/day may be regarded as a 'No Observed Adverse Effect Level' (NOAEL) for animals of either sex. The No Observed Effect Level (NOEL) was considered to be 10 mg/kg bw/day for males and 100 mg/kg bw/day for females.

In summary, reproductive function was not affected by the test substance administration as evident from the results of developmental toxicity studies. NOAEL for maternal toxicity was established at the same levels for general systemic toxicity and developmental toxicity. No test substance-related influence was noted on the reproductive parameters of the dams at all dose levels tested in three developmental studies. Therefore, NOAEL for reproductive toxicity is covered by NOAEL of the developmental toxicity studies for a corresponding route of exposure. In the 90-day study, no adverse effects were detected during the oestrous cycle assessment and semen parameters assessment. There were no abnormalities detected in reproductive organs, their weights and histopathology. Based on the results of the 90-day study, the highest dose level of 500 mg/kg bw may be regarded as NOAEL for reproductive function.

Effects on developmental toxicity

Description of key information

- Inhalation developmental toxicity study (OECD 414): Crl:CD(SD) rats, whole body; conc.: 0, 0.3, 0.6 and 1.2 mg/L (corresponding to 76.2, 152.6, and 315.8 mg/kg/day, respectively); GD 6-19; NOAEC: 0.6 mg/L (152.6 mg/kg bw).

- Dermal developmental study (OECD 414): Crl:CD(SD) rats, dermal daily exposure for approximately 23 hours each day; dose levels: 0, 375, 500 and 750 mg/kg bw; GD: 6-19; NOAEL: 500 mg/kg bw.

- Oral developmental study (OECD 414): Crl:CD(SD) rats, gavage; dose levels: 200, 300, 400 and 500 mg/kg bw; GD: 6-19; NOAEL: 400 mg/kg bw.

- Supporting oral developmental study (OECD 414): CD rats; gavage; dose levels: 0, 5, 50 and 500 mg/kg bw; NOAEL > 500 mg/kg bw for foetal organisms.

- Oral Rabbit developmental toxicity study according to OECD 414. Developmental NOAEL of 300 mg/kg/day, the highest dose tested. No developmental effects found.

Since developmental toxicity is the endpoint of concern by structurally related analogues N-methylpyrrolidone and N-ethylpyrrolidone, NOAELs from developmental toxicity studies will be used for hazard assessment (DNEL derivation). Fertility effects and general toxicity are covered by the developmental NOAELs.

Remark

Additional link to relevant study: ESR contained in section 13 of the IUCLID file

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-19 to 2016-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at room temperature under a nitrogen blanket, and was considered stable under these conditions.
- Stability under test conditions: Documentation regarding the purity and stability of the test substance is on file with the Sponsor and Charles River.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: No
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 75 days old upon receipt; The selected females were approximately 12 weeks old when paired for breeding.
- Weight at study initiation: Body weight values of the selected females ranged from 230 g to 284 g on Gestation Day 0.
- Fasting period before study: no
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH).
- Diet (e.g. ad libitum): ad libitum except during exposure periods (The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River).
- Water (e.g. ad libitum): ad libitum except during exposure periods (Reverse osmosis-purified (on-site) drinking water)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Non-exposure periods: 68°F to 78°F (20°C to 26°C); actual mean daily temperature ranged from 72.3°F to 74.1°F (22.4°C to 23.4°C) during the study. Exposure periods: The mean temperature was between 19°C to 25°C.
- Humidity (%): Non-exposure periods: 30% to 70%; actual mean daily relative humidity ranged from 45.4% to 59.0% during the study. Exposure periods: The mean relative humidity was between 30% to 70%.
- Air changes (per hr): Non-exposure periods: 10. Exposure periods: All chambers were operated under dynamic conditions, at least 12-15 air changes per hour, at a slight negative pressure.
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- Oxygen content was measured during the method development phase and was 20.9% for Groups 2-4.

IN-LIFE DATES: From: 02 Aug 2016 (animal receipt) To: 02 Sep 2016 (last laparohysterectomy).
Route of administration:
inhalation: mixture of vapour and aerosol / mist
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.4 - <= 2.7 µm
Geometric standard deviation (GSD):
1.73
Remarks on MMAD:
The target range for MMAD is 1.0 to 3.0 microns and GSD is 1.5 to 3.0. Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Mean MMAD and GSD were within the target values (table 10). MMAD values were 2.7, 2.4 and 2.4 for the substance-treated groups 2, 3 and 4, respectively. GSD were of 1.81, 1.78 and 1.73 for the substance-treated groups 2, 3 and 4, respectively.
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: Exposures were conducted using four 1000-L glass and stainless steel whole-body inhalation exposure chambers. One exposure chamber was dedicated for each group for the duration of the study.

- Method of holding animals in test chamber: For each day’s exposure, the animals were transferred to exposure caging in the colony room, transported to the exposure room, exposed for the requisite duration, and returned to their home cages in the animal colony room. The animal cage batteries were rotated on a daily basis between the 3 battery positions within the chamber to help ensure a similar exposure for all animals within each group over the duration of the exposure period.

- Source and rate of air: Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source.

- Method of conditioning air: not reported.

- System of generating particulates/aerosols: An individual generation system was used for each test substance exposure chamber. A mixed liquid aerosol/vapor atmosphere of the test substance was generated as follows. During generation trials as part of the range-finding study, it was noted that the test substance was not compatible with a plastic clinical nebulizer, but it was compatible with the glass jar of a Collison nebulizer. The test substance was aerosolized using 1 or more Collison nebulizers. A 6-jet nebulizer was used for Chamber 2 and Chamber 3, and two 6-jet nebulizers were used for Chamber 4. Using a regulator, breathing quality compressed air from the facility in-house air source was delivered to each nebulizer. Test substance was delivered to the nebulizers through the use of an infusion pump.
For Chamber 4, the resulting liquid aerosol/vapor atmosphere from the 6-jet nebulizers were combined at a 4-way ‘cross’-fitting where it was mixed with dry, compressed air. For Chambers 2 and 3, the resulting liquid aerosol/vapor atmosphere from the respective nebulizer was mixed with additional dry, compressed air at a ‘T’-fitting. The amount of compressed air for Chambers 2, 3, and 4 were controlled using a regulator.
In order to produce an exposure atmosphere with an appropriate particle-size, the resulting liquid aerosol/vapor mixture for all test substance chambers was first directed through a glass cyclone in order to reduce the particle-size to under 3 microns. The output from each cyclone was delivered to the chamber inlet, where it was mixed with supply air to achieve the desired target concentration of the test substance.

- Temperature, humidity, pressure in air chamber: Chamber ventilation rate and negative pressure within the chambers were continually monitored and recorded approximately every 45 minutes during the 6-hour exposure periods. Temperature and relative humidity within each exposure chamber were determined at least 3 times during each period. The mean temperature and mean relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively. Oxygen content was measured during the method development phase and was 20.9% for Groups 2-4.

- Air flow rate: The airflow rates for each whole-body chamber were monitored by measuring the pressure drop between the ports of an orifice plate using a Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge was calibrated for conversion from pressure to airflow in standard liters per minute through the use of a Fox Gas Mass Flowmeter Transmitter (model no. FT2, Fox Thermal Instruments; Marina, CA). Sample flow through the sample train was controlled using a needle valve connected to the facility vacuum source. Prior to each sample collection, the sample flow rate was measured using a Mini-Buck Calibrator (model no. M-5, A.P Buck Inc.; Orlando, FL). An approximate sample flow rate was 2.0 L/min for 8, 2.5, and 1.5 minutes for Chambers 2, 3, and 4, respectively. Chamber ventilation rate (airflow) and negative pressure within each exposure chamber were continually monitored and recorded at approximately 45-minute intervals through the use of the Inhalation Toxicology Data Acquisition System (WINH) and a personal computer.

- Air change rate: All chambers were operated under dynamic conditions, at least 12-15 air changes per hour, at a slight negative pressure.

- Method of particle size determination: Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Aerosol particle-size measurements were conducted using a 7-stage stainless-steel cascade
impactor (model no. 02-100-2L, IN-TOX Products; Moriarty, NM). Pre-weighed, 22-mm stainless-steel collection substrates were used for stage 1 to 7. A pre-weighed, 25-mm glass fiber filter (Type A/E, PALL Corporation) was used as the final collection substrate. Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Samples of the mixed liquid aerosol/vapor atmosphere of test substance were collected at an approximate sample flow rate of 2.0 LPM for 8, 2.5, and 1.5 minutes for Chambers 2, 3, and 4, respectively. Sample flow rates were measured using a Mini-Buck Calibrator (model no. M-5). Following sample collection, the aerosol retained on the filters was determined by re-weighing the filters and the particle-size was calculated based on the impactor stage cut-offs. The particle-size was expressed as the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD).

- Treatment of exhaust air: All test substance atmosphere chamber exhaust passed through an activated-carbon drum prior to passing through the facility exhaust system, consisting of redundant exhaust blowers preceded by activated-charcoal and HEPA-filter units.

TEST ATMOSPHERE
- Brief description of analytical method used: Following each sample collection, aerosol content was measured using standard gravimetric methods and the vapor concentration was determined using analytical techniques (liquid-phase injection gas chromatograph (GC) with flame ionization detection). The filter was re-weighed and the aerosol concentration (mg/L) was calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume.
- Samples taken from breathing zone: yes (the description please see below in "Details on analytical verification of doses or concentrations".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal exposure concentrations were calculated for each test substance exposure chamber from the total amount of test substance used during each generation period and the total volume of air that passed through the chamber during exposure. Test substance usage was determined by weighing pre-filled syringes prior to and at the termination of each generation. Total air volume was calculated by multiplying the daily mean ventilation rate by the duration of generation.

Samples of the exposure atmospheres were collected using a sampling train consisting of a pre-weighed, glass fiber filter in-line with a 30-mL midget impinger at approximately 60-minute intervals. The filter was held in a filter holder and placed in the animal-breathing zone of the chamber. The impinger was filled with approximately 10 mL of isopropanol as the trapping liquid. Prior to each sample collection, the sample flow rate was measured using a calibrator.
Aerosol concentrations
Following each sample collection, aerosol content was measured using standard gravimetric methods and the vapor concentration was determined using analytical techniques. The filter was re-weighed and the aerosol concentration (mg/L) was calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume.
Vapor Concentrations
Analyzed concentrations of the test substance vapor in the exposure atmospheres were determined using a liquid-phase injection gas chromatograph (GC) with flame ionization detection.
Following sample collection, a portion of the impinger liquid was transferred to a 2-mL vial and analyzed using a GC. Liquid sample injection onto the chromatography column occurred via manual injection (approximately 2 μL) into a split/splitless injector, the chromatograph was displayed, and the area under the sample peak was calculated and recorded. Test substance concentration within the impinger liquid, in μg/mL, was calculated using the ln-quadratic equation based on the GC calibration curve. The total amount of vaporized test substance recovered was determined by multiplying the impinger liquid concentration by the impinger solvent volume (10 mL). The vapor concentration was determined by dividing the amount of test substance recovered by the sample volume.
Total Test Substance Concentrations
Total test substance (combined aerosol and vapor) was calculated by addition of the gravimetrically and analytically determined concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females.
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: not reported.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
6 hours
Frequency of treatment:
once daily
Duration of test:
during Gestation Days 6–19.
Dose / conc.:
0.3 mg/L air (nominal)
Remarks:
Mean analyzed exposure concentrations was 0.29 mg/L and corresponded to mean estimated inhaled (delivered) dosage level 76.2 mg/kg/day.
Dose / conc.:
0.6 mg/L air (nominal)
Remarks:
Mean analyzed exposure concentrations was 0.58 mg/L and corresponded to mean estimated inhaled (delivered) dosage level 152.6 mg/kg/day.
Dose / conc.:
1.2 mg/L air (nominal)
Remarks:
The highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). The mean analyzed exposure concentration was 1.2 mg/L and corresponded to mean estimated inhaled (delivered) dosage levels of 315.8 mg/kg/day.
No. of animals per sex per dose:
24
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: 2-week range-finding study.
In that study, the test substance was administered via whole-body inhalation exposure, 6 hours per day, for 14 consecutive days to 3 groups of 5 female Crl:CD(SD) rats. As part of the generation trials for this study, attempts were made to produce a maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). It was estimated that the highest exposure concentration that met this criteria would be 1.0 mg/L. Therefore, target exposure concentrations were 0.25, 0.5, and 1.0 mg/L for Groups 2, 3, and 4, respectively. Mean exposure concentrations (total test substance; aerosol plus vapor) over the duration of the exposure period were 0.29, 0.64, and 1.23 mg/L for the same respective groups. Minor adjustments to generation settings (nebulizer pressure and/or dilution airflow) allowed the actual achieved concentration to be approximately 1.2 mg/L and consistently maintain a particle size under 3 microns (achieved 2.6 microns).
All animals survived to the scheduled necropsy. There were no test substance-related effects on body weights, food consumption, macroscopic pathology, or lung weights in any of the test substance-exposed groups. Clinical observations noted in the 1.0 mg/L group included partial closure of the right and left eyes, impaired equilibrium, and increased respiration rate. Test substance-related clinical observations of rales were noted in the 0.5 and 1.0 mg/L groups. Observations of rales were noted beginning as early as Study Day 3 in the 1.0 mg/L group and continued through the remainder of the study period. An animal in the 0.5 mg/L group was observed with rales beginning on Study Day 4.
Based on the lack of dose-limiting toxicity observed at up to 1.2 mg/L, exposure concentrations of 0.3, 0.6, and 1.2 mg/L were chosen for this study. The selected route of administration for this study was inhalation exposure because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program, which randomized the animals based on stratification of the Gestation Day 0 body weights in a block design. Animals not assigned to study were transferred to the Charles River rat colony.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily during Gestation Days 0–20 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at approximately the midpoint of each 6-hour exposure and approximately 1 hour following exposure. The absence or presence of findings was recorded for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on Gestation Days 0 and 6–20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for Gestation Days 6–9, 9–12, 12–15, 15–20, and 6–20.
Gravid uterine weight was collected and net body weight (the Gestation Day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Individual food consumption was recorded on Gestation Days 0 and 6–20 (daily).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. Wet lung weights were recorded for all females at the scheduled necropsy, and the lungs were subsequently placed in 10% neutral-buffered formalin for possible future histopathological examination. Other maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings; representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification.

- External examinations: Yes: all per litter. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.

- Soft tissue examinations: Yes: all per litter. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development.

- Skeletal examinations: Yes: all per litter. Following fixation in alcohol, each fetus was stained with Alizarin Red S7 and Alcian Blue8. Fetuses were then examined for skeletal malformations and developmental variations.

- Head examinations: Yes: all per litter. Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Statistics:
All statistical tests were performed using WTDMS™. Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group. Each mean was presented with the S.D., S.E., and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, S.D. and S.E. on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, lung weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-exposed groups to the control group.
Indices:
Gestation Day 20 Laparohysterectomy
Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group)/ No. Gravid Females/Group

2. Proportional Litter Basis:
Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group;

Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter)/ No. Implantation Sites/Litter x 100

Fetal Morphological Examination
The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%))/ No. Litters/Group

Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter)/ No. Viable Fetuses/Litter x 100
Historical control data:
Charles River has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat (Number of Datasets in Historical Control: 79-80). This animal model has been proven to be susceptible to the effects of developmental toxicants.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, increased incidences of red material around the nose, mouth, and forelimbs was noted in the 0.6 and 1.2 mg/L groups generally throughout the treatment period; these observations were noted primarily at approximately 1 hour following exposure at 0.6 mg/L and at the daily examinations and approximately 1 hour following exposure at 1.2 m/L. The aforementioned observations were considered adverse at 1.2 mg/L; because these findings were generally resolved by the following daily examination in the 0.6 mg/L group, they were considered nonadverse at this exposure level. In the 1.2 mg/L group, an increased incidence of clear material on the ventral neck was noted primarily during Gestation Days 16–20 at the daily examinations and/or approximately 1 hour following exposure and was considered test substance-related and adverse. No other test substance-related clinical observations were noted at the daily examinations, midpoint of exposure, or approximately 1 hour following exposure at any exposure level. Observations noted in the exposed groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.
Mortality:
no mortality observed
Description (incidence):
All females in the control, 0.3, 0.6, and 1.2 mg/L groups survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 1.2 mg/L group, test substance-related mean body weight losses or lower mean body weight gains were noted throughout the exposure period (Gestation Days 6–9, 9–12, 12–15, and 15–20) and resulted in a lower mean body weight gain when the entire exposure period (Gestation Days 6–20) was evaluated compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). In addition, mean body weights in this group were lower (5.4% to 11.5%; significant at p < 0.01) than the control group during Gestation Days 8–20. Lower mean net body weight and net body weight gain were noted in the 1.2 mg/L group compared to the control group; differences were significant (p < 0.01). In addition, a significantly (p < 0.01) lower mean gravid uterine weight was noted in the 1.2 mg/L group compared to the control group. The aforementioned effects noted in the 1.2 mg/L group were considered adverse.
In the 0.3 and 0.6 mg/L groups, significantly (p < 0.01) lower mean body weight gains were noted during Gestation Days 6–9 compared to the control group. Mean body weight gains in these groups were similar to the control group for the remainder of the exposure period (Gestation Days 9–12, 12–15, and 15–20). Due the decrements in mean body weight gain noted at the beginning of the exposure period, a significantly (p < 0.01) lower mean body weight gain was noted in the 0.6 mg/L group compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated; mean body weight gain in the 0.3 mg/L group was similar to the control group for this interval. The lower mean body weight gains noted in the 0.3 and 0.6 mg/L groups were not of sufficient magnitude to affect mean body weights at these exposure levels, and therefore were considered nonadverse. Mean maternal net body weights, net body weight gains, and gravid uterine weights in the 0.3 and 0.6 mg/L groups were unaffected by test substance exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, lower mean maternal food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 1.2 mg/L group throughout the exposure period (Gestation Days 6–9, 9–12, 12–15, and 15–20) and resulted in lower mean food consumption in this group compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated; differences were significant (p < 0.01). The lower mean food consumption in the 1.2 mg/L group correlated with the mean body weight losses and lower mean body weight gains and was considered adverse.
Test substance-related, lower mean food consumption was noted during Gestation Days 6–9 in the 0.3 mg/L group and during Gestation Days 6–9, 9–12, and 12–15 in the 0.6 mg/L group compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). Mean food consumption was similar to the control group during Gestation Days 9–12, 12–15, and 15–20 at 0.3 mg/L and during Gestation Days 15–20 at 0.6 mg/L. As a result of the initial decrements in mean food consumption, significantly (p < 0.05 or p < 0.01) lower mean food consumption was noted in these groups compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated. However, the differences noted in the 0.3 and 0.6 mg/L groups were not of sufficient magnitude to affect mean body weights in these groups, and therefore were considered nonadverse.
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
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 test substance-related effects on mean lung weights were noted in the 0.3, 0.6, and 1.2 mg/L groups. Differences from the control group were slight and not statistically significant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on Gestation Day 20, no test substance-related internal findings were observed at exposure levels of 0.3, 0.6, and 1.2 mg/L. Macroscopic findings observed in the test substance-exposed groups occurred infrequently and/or in a manner that was not exposure-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. Parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
see above
Early or late resorptions:
no effects observed
Description (incidence and severity):
see above
Dead fetuses:
no effects observed
Description (incidence and severity):
see above
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of 2 and 1 females in the control and 0.6 mg/L groups, respectively, all females were determined to be gravid.
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
0.6 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male, female, and combined fetal weights in the 1.2 mg/L group (3.5 g, 3.3 g, and 3.4 g, respectively) were significantly (p < 0.01) lower (approximately 13%) than the control group values (4.0 g, 3.8 g, and 3.9 g, respectively) and were also below the minimum mean values in the Charles River Ashland historical control data (version 2016.03; 3.682 g, 3.458 g, and 3.573 g, respectively). The lower mean fetal weights at 1.2 mg/L correlated with decrements in maternal mean body weight gain and food consumption noted in this group during the fetal period (Gestation Days 15–20), which is the time of greatest fetal growth, and were considered adverse.
Lower (approximately 5%) mean male, female, and combined fetal weights were noted in the 0.3 and 0.6 mg/L groups; differences were generally significant (p < 0.05 or p < 0.01). However, the values at 0.3 and 0.6 mg/L (3.8 g for males, 3.6 g for females, and 3.7 g for combined) were greater than the minimum mean values and only 2% to 3% lower than the mean (3.898 g for males, 3.697 g for females, and 3.801 g for combined) and median (3.883 g for males, 3.696 g for females, and 3.792 g for combined) values within the Charles River Ashland historical control data. The concurrent control group values for fetal body weight (4.0 g for males, 3.8 g for females, and 3.9 g for combined) were 2% to 3% heavier (approximately equal to the 75th quartile; 3.971 g for males, 3.761 g for females, and 3.861 g for combined) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights in the 0.3 and 0.6 mg/L groups were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups. The absence of a dose-response relationship between the 0.3 and 0.6 mg/L group fetuses also suggests a lack of effect at these exposure concentrations. In addition, the magnitude of change in these groups compared to the control group was not as severe as the difference in the 1.2 mg/L group. Therefore, the lower mean fetal weights at 0.3 and 0.6 mg/L were not considered test substance-related.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. Parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see above
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see above
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological evaluation were 323(22), 358(24), 339(23), and 350(24) in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. Malformations were observed in 2(2), 2(2), 1(1), and 4(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.
No test substance-related external malformations were observed for fetuses at any exposure level. Fetal anasarca was noted for 1 fetus each in the control (No. 6130-16) and 0.3 (No. 6082-07) mg/L groups. In the 0.3 mg/L group, Fetus No. 6094-16 was noted with anophthalmia (bilateral; confirmed skeletally as orbits smaller than normal). The aforementioned malformations were noted in single fetuses, similarly in the control group, and were not observed in an exposure-related manner; therefore, they were not considered test substance-related.
No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related skeletal malformations were noted for fetuses at any exposure level. In the 1.2 mg/L group, 2 fetuses (Nos. 6025-10 and 6113-02) were noted with costal cartilage anomaly (bifurcated or fused costal cartilage) and 1 fetus (No. 6113-07) was noted with 8 cervical vertebrae (with normal presacral vertebrae count). Fetus No. 6065-03 in the 1.2 mg/L group was noted with severely malaligned sternebrae; this finding was also noted for 1 fetus (No. 6089-14) in the control group. Sternoschisis (no. 4 or no. 6 sternal bands not joined) was noted for 1 fetus each in the control (No. 6130-16) and 0.6 (No. 6076-04) mg/L groups. The aforementioned skeletal malformations were observed in single fetuses, similarly in the control group, were not observed in an exposure-related manner, and/or the differences in the mean litter proportions were not statistically significantly different from the concurrent control group and the values were within the ranges of the Charles River Ashland historical control data; therefore, they were not considered test substance-related.
Test substance-related skeletal developmental variations were noted in the 1.2 mg/L group. In the 1.2 mg/L group, a significantly (p < 0.01) lower mean litter proportion of cervical centrum no. 1 ossified was noted compared to the control group (3.4% versus 27.3% per litter). In addition, a higher (not statistically significant) mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified was noted in the 1.2 mg/L group (23.1% per litter) compared to the control group (9.4% per litter); the value at 1.2 mg/L exceeded the maximum mean value in the Charles River Ashland historical control data (19.86% per litter). The aforementioned findings noted at 1.2 mg/L were considered indicators of developmental delay and correlated with the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level.
No other test substance-related skeletal developmental variations were noted at any exposure level. Other findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test substance-related visceral malformations were observed for fetuses at any exposure level. In the 0.3 mg/L group, Fetus No. 6094-16 was noted with an interventricular septal defect (a <1 mm in diameter opening in the anterior portion of the septum); this fetus was also noted with an external malformation. Because this finding was noted in a single fetus and did not occur in an exposure-related manner, this finding was not considered test substance-related. In the control group, Fetus No. 6130-16 was noted with a bulbous aorta (ascending and aortic arches).
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 2 (Nos. 6020-12 and 6071-13), 1 (No. 6095-14), 1 (No. 6047-06), and 1 (No. 6110-13) fetuses in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred infrequently, at similar frequencies in the control group, and in a manner that was not exposure-related.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 323(22), 358(24), 339(23), and 350(24) in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. Malformations were observed in 2(2), 2(2), 1(1), and 4(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations, when observed in the test substance-exposed groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in an exposure-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations were attributed to the test substance.
Test substance-related fetal skeletal developmental variations were noted in the 1.2 mg/L group. A higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and a lower mean litter proportion of cervical centrum no. 1 ossified were noted in the 1.2 mg/L group. These findings were considered secondary to the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level. No other test substance-related developmental variations were noted at any exposure level.
Key result
Dose descriptor:
NOAEL
Effect level:
0.6 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: Test substance-related fetal skeletal developmental variations
Key result
Abnormalities:
no effects observed
Localisation:
skeletal: sternum
Description (incidence and severity):
Variations: In the 1.2 mg/L group, a test substance-related higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified were noted compared to the control group; these findings were considered indicators of developmental delay and correlated with the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1.2 mg/L air (nominal)
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:
not specified

Table 4. Overall Nominal Exposure Concentrations

Exposure Chamber: 2 3 4
Target Concentration (mg/L): 0.30 0.60 1.2
Nominal Concentration (mg/L): 1.0 1.8 4.9
Standard Deviation: 0.11 0.12 1.13
N: 17 17 17

The overall mean nominal concentrations for each exposure group are presented below. Due to the use of glass cyclones, a large amount of liquid test substance was removed from the generation atmosphere prior to entering the exposure chamber. This resulted in a greater calculated nominal concentration.

The overall mean analyzed exposure concentrations for each group are presented below:

Table 5. Overall Mean Exposure Concentrations

Exposure Chamber: 1 2 3 4
Target Concentration (mg/L): 0 0.3 0.6 1.2
Mean Concentration (mg/L): 0 0.29 0.58 1.2
Standard Deviation: 0.0 0.031 0.035 0.05
N: 5 17 17 17

The overall mean aerosol particle size for each test substance-treated group is presented below:

Table 6. Mean Aerosol Particle-Size

Exposure Chamber: 2 3 4
Mean MMAD (microns): 2.7 2.4 2.4
Mean GSD: 1.81 1.78 1.73
N: 3 3 3

The mean estimated inhaled (delivered) dose and the mean deposited dose for each interval and the overall exposure period are presented below. The deposited dose will be estimated at 10% of the estimated inhaled dose.

Table 7. Mean Estimated Delivered Dosage

Exposure Chamber: 1 2 3 4
Target Concentration (mg/L): 0 0.3 0.6 1.2
Mean Concentration (mg/L): 0 0.29 0.58 1.2
Females (mg/kg/day): 0.0 76.2 152.6 315.8
Deposited Dose (mg/kg/day): 0.0 7.62 15.26 31.58
Conclusions:
Target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines).
Maternal toxicity was evidenced by adverse clinical observations and mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption in the 1.2 mg/L group throughout the gestation treatment period. Developmental effects were noted at 1.2 mg/L as evidenced by lower mean fetal weights, which correlated with a lower mean gravid uterine weight and decrements in maternal food consumption and body weight gain during the fetal period (Gestation Days 15-20). Lower mean fetal weights were also noted in the 0.3 and 0.6 mg/L groups; however, mean fetal weight values in these groups were greater than the minimum mean values and only slightly lower than the mean and median values in the Charles River Ashland historical control data. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups, and were not considered test substance-related. Test substance-related fetal skeletal developmental variations (higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified) were noted in the 1.2 mg/L group, which corresponded with the lower mean fetal weights noted in this group. No adverse maternal or developmental effects were noted at 0.3 and 0.6 mg/L. Based on these results, an exposure level of 0.6 mg/L was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance was administered via whole-body inhalation exposure to bred Crl:CD(SD) rats. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively.
Executive summary:

The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. The test substance was administered via whole-body inhalation exposure to 3 groups of 24 bred female Crl:CD(SD) rats for 6 hours per day from Gestation Days 6–19. Based on a range-finding study in nonpregnant female rats, target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). Mean analyzed exposure concentrations were 0.29, 0.58, and 1.2 mg/L for the same respective groups. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively. A concurrent control group composed of 24 bred females were exposed to humidified, filtered air on a comparable regimen. The females were approximately 13 weeks of age at the initiation of exposure. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On Gestation Day 20, a laparohysterectomy was performed on each female and the lungs were weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 0.3, 0.6, and 1.2 mg/L groups survived to the scheduled necropsy. Test substance-related, increased incidences of red material around the nose, mouth, and forelimbs were noted in the 0.6 and 1.2 mg/L groups generally throughout the treatment period at the daily examinations and approximately 1 hour following exposure and was considered adverse at 1.2 mg/L; these observations were generally resolved by the following daily examination in the 0.6 mg/L group, and were therefore considered nonadverse at this exposure level. In the 1.2 mg/L group, a test substance-related increased incidence of clear material on the ventral neck was noted during the latter half of gestation at the daily examinations and/or approximately 1 hour following exposure and was considered adverse. No other test substance-related clinical observations were noted at the daily examinations, midpoint of exposure, or approximately 1 hour following exposure at any exposure level. In the 1.2 mg/L group, test substance-related mean body weight losses or lower mean body weight gains with corresponding reductions in mean food consumption were noted throughout the exposure period and resulted in a lower mean body weight gain and food consumption when the entire exposure period (Gestation Days 6–20) was evaluated compared to the control group. In addition, mean body weights in this group were 5.4% to 11.5% lower than the control group during Gestation Days 8-20. Test substance-related lower mean net body weight, net body weight gain, and gravid uterine weight were also noted in the 1.2 mg/L group. The aforementioned body weight effects noted in the 1.2 mg/L group were considered adverse. In the 0.3 and 0.6 mg/L groups, lower mean body weight gains were noted during Gestation Days 6–9 compared to the control group; mean body weight gains in these groups were similar to the control group for the remainder of the exposure period. Lower mean food consumption was noted during Gestation Days 6–9 in the 0.3 mg/L group and during Gestation Days 6–15 in the 0.6 mg/L group compared to the control group and resulted in lower mean food consumption in these groups when the entire treatment period (Gestation Days 6–20) was evaluated. However, the aforementioned differences in body weight gain and food consumption noted at 0.3 and 0.6 mg/L were not of sufficient magnitude to affect mean body weights at these exposure levels, and therefore were considered test substance-related but nonadverse. Mean maternal body weights, net body weights, net body weight gains, and gravid uterine weights in the 0.3 and 0.6 mg/L groups were unaffected by test substance exposure. No test substance-related macroscopic findings or changes in mean lung weights were noted for females at any exposure level at the scheduled necropsy on Gestation Day 20.

Mean fetal weights (male, female, and combined) in the 0.3, 0.6, and 1.2 mg/L groups were up to 5.3%, 5.3%, and 13.2% lower, respectively, than the control group. The differences in mean fetal weights noted in the 1.2 mg/L group correlated with decrements in maternal mean body weight gain and food consumption noted in this group during the fetal period (Gestation Days 15–20), which is the time of greatest fetal growth, and were considered adverse. When compared to the Charles River Ashland historical control data, the mean fetal weight values in the 0.3 and 0.6 mg/L groups were greater than the minimum mean values and only slightly lower than the mean and median values. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups. Additionally, the magnitude of change in these groups compared to the control group was not as severe as the difference in the 1.2 mg/L group and there was no dose-response relationship between the 0.3 and 0.6 mg/L groups. Therefore, the lower mean fetal weights at 0.3 and 0.6 mg/L were considered unrelated to treatment. Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. A test substance-related, higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and a lower mean litter proportion of cervical centrum no. 1 ossified was noted in the 1.2 mg/L group compared to the control group. The aforementioned findings noted at 1.2 mg/L were considered indicators of developmental delay and secondary to the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level. There were no test substance-related malformations noted at 0.3, 0.6, and 1.2 mg/L or developmental variations noted at 0.3 and 0.6 mg/L.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May 2016 to 10 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
the United States EPA GLP Standards 40 CFR Part 160 and 40 CFR Part 792 (l 6-0ct-1989 and l 8-Sep-1989, respectively) and the OECD Principles of GLP [C(97) 186/Final] (26-Nov-1997).
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test substance was stored at room temperature under a nitrogen blanket, and was considered stable under these conditions.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance in the vehicle was previously established to be stable and homogeneous following at least 10 days of refrigerated and room temperature storage under a nitrogen blanket at concentrations of 40 to 200 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: preparation of test substance formulations in vehicle.
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: [Crl:CD(SD)]
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 79 days old upon receipt. The selected females were approximately 12 weeks old when paired for breeding.
- Weight at study initiation: The day following receipt, all animals were weighed and clinical observations were recorded. Body weight values ranged from 214 g to 268 g on gestation day 0.
- Fasting period before study: no
- Housing: Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- Diet (e.g. ad libitum): ad libitum; The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River.
- Water (e.g. ad libitum): ad libitum; Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system.
- Acclimation period: 14 days. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behavior. All animals were acclimated to the Elizabethan collars during the acclimation period according to Charles River SOPs.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 73°F ± 5°F (23°C ± 3°C). Actual mean daily temperature ranged from 72.3°F to 72.5°F (22.4°C to 22.5°C) during the study.
- Humidity (%): 50% ± 20%. Actual mean daily relative humidity ranged from 39.5% to 55.8% during the study.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 May 2016 (Experimental starting date (animal receipt)) To: scheduled necropsy on gestation day 20
Route of administration:
dermal
Vehicle:
water
Remarks:
deionized water (prepared on-site)
Details on exposure:
TEST SITE
- Area of exposure: Prior to the first day of dose administration, and as often as needed thereafter, the hair was clipped in a rectangular shape from the dorsal scapular area of each animal; a different set of clippers was used for control and test substance-treated animals to avoid potential cross-contamination. Care was taken not to abrade the skin.
The formulations were applied over the center of the clipped area and distributed to avoid running; the application site was approximately 10% of the total body surface area. The four corners of the application site were marked with indelible ink to allow proper identification of the treated and untreated skin.
- % coverage: 10
- Type of wrap if used: no, the treated animals were outfitted by the Elizabethan collars to restrict access to the dermal application site and avoiding grooming
- Time intervals for shavings or clipplings: Prior to the first day of dose administration, and as often as needed thereafter.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no. Following approximately 23 hours of exposure, the application sites were gently patted with a disposable paper towel to remove residual test substance.
- Time after start of exposure: 23 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
- Concentration (if solution): 75, 100 and 150 mg/mL for dose levels of 375, 500 and 750 mg/kg bw, respectively (see table 2).
- Constant volume or concentration used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2°C to 8°C), purged with nitrogen. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

The test substance in the vehicle was previously established to be stable and homogeneous following at least 10 days of refrigerated and room temperature storage under a nitrogen blanket at concentrations of 40 to 200 mg/mL.
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last 75, 100, and 150 mg/mL dosing formulations and from the middle stratum of the first and last control dosing formulations. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of an aliquot taken from the first 75 and 150 mg/mL dosing suspensions following refrigerated (2°C to 8°C), purged with nitrogen storage for 9 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored refrigerated (2°C to 8°C) under a nitrogen blanket as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
Details on mating procedure:
- Impregnation procedure: cohoused
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained.
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: not reported
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
single application
Frequency of treatment:
once daily
Duration of test:
from gestation days 6 through 19.
Dose / conc.:
375 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Based on the number of animals available for replacement, the total number of pregnant females assigned to the control, 375, 500, and 750 mg/kg/day groups was 24, 23, 24, and 24, respectively.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous dose range-finding dermal study in rats. In that study, pregnant rats were dosed from gestation days 6-19 at dosage levels of 0, 250, 500, and 750 mg/kg/day. There were no deaths that were considered related to treatment. Lower mean body weight gains were observed in the 750 mg/kg/day group when the overall treatment period was evaluated (gestation days 6-20). In addition, lower mean fetal weights were noted in the 750 mg/kg/day group compared to the control group. As a result, dosage levels of 375, 500, and 750 mg/kg/day were selected for the current study. The high dosage level was expected to produce some evidence of maternal toxicity without resulting in mortality.
- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design.
- Other: Replacement animals were arbitrarily assigned based on body weight. On 13 Jun 2016 (gestation day 6), 23 females were dosed but did not have proper access to food because they were provided the incorrect food jars for use with Elizabethan collars. This resulted in severe body weight loss and reduced food consumption for these females from gestation day 6 to 7. Therefore, these females were removed from study on 14 Jun 2016 and 18 additional females were assigned to the study. Based on the number of animals available for replacement, the total number of pregnant females assigned to the control, 375, 500, and 750 mg/kg/day groups was 24, 23, 24, and 24, respectively.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration and to confirm the Elizabethan collar remained on the animal approximately 3 hours following each dose administration. The absence or presence of findings was recorded for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded.
Wet lung weights were recorded for all females at the scheduled necropsy. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

OTHER: Dermal Observations:
The application site was scored daily (prior to dose administration during the treatment period) for erythema and oedema in accordance with a 4-step grading system of very slight, slight, moderate, and severe. Other remarkable dermal findings, if present, were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes
Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group))/ No. Gravid Females/Group

2. Proportional Litter Basis:
Summation Per Group (%) = (Sum of Postimplantation Loss/Litter (%)) / No. Litters/Group

Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter) / No. Implantation Sites/Litter x 100
Fetal examinations:
- External examinations: Yes: all per litter. Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the Charles River study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded.
- Soft tissue examinations: Yes: all per litter. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development.
- Skeletal examinations: Yes: all per litter. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- Head examinations: Yes: all per litter. Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the S.D., S.E., and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, S.D., and S.E. on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, organ weights, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
Indices:
The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%)) / No. Litters/Group
Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter) / (No. Viable Fetuses/Litter) x 100
Historical control data:
WIL Research has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A slightly increased incidence of red material around the eyes was noted for 7, 11, and 12 females in the 375, 500, and 750 mg/kg/day groups, respectively, generally throughout the treatment period at the daily examinations; however, given the low occurrence of this finding (generally 1-4 occurrences per animal) and the inability of the animals to groom while outfitted with Elizabethan collars, this finding was considered nonadverse. No other test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Other findings noted in the treated groups, including red material around the nose and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
No dermal observations were noted for females in the control, 375, 500, and 750 mg/kg/day groups.
Mortality:
no mortality observed
Description (incidence):
All females in the control, 375, 500, and 750 mg/kg/day groups survived to the scheduled necropsy on gestation day 20.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight losses were noted in all groups, including the control group, following the initiation of dose administration (gestation day 6-7); these differences were likely due to the placement of the Elizabethan collars. In the 750 mg/kg/day group, a test substance-related mean body weight loss (not statistically significant) was noted during gestation days 6-9 followed by mean body weight gains that were similar to the control group during gestation days 9-12 and 12-15. Mean body weight gains in this group were lower (23.5% to 37.5%) during gestation days 17-19 and resulted in a lower mean body weight gain for the gestation days 15-20 interval compared to the control group; differences were significant (p<0.05 or p<0.01). The decrements in mean body weight gain noted in the 750 mg/kg/day group resulted in a significantly (p<0.01) lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated compared to the control group. In addition, mean body weights in the 750 mg/kg/day group were significantly (p<0.05 or p<0.01) lower (up to 7.4%) compared to the control group during gestation days 8-20. The aforementioned body weight effects noted in the 750 mg/kg/day group were considered adverse. A test substance-related lower mean net body weight and net body weight gain were noted in the 750 mg/kg/day group compared to the control group and were considered adverse; differences were significant (p<0.01). In addition, a test substance-related, significantly (p<0.01) lower mean gravid uterine weight was noted in the 750 mg/kg/day group.
In the 375 and 500 mg/kg/day groups, mean body weight gains were generally similar to the control group for the remainder of the treatment period (gestation days 6-9, 9-12, 12-15, 15-20, and 6-20). A significantly (p<0.01) lower mean body weight gain was noted at 500 mg/kg/day compared to the control group on gestation day 17-18; however, this difference was transient and not of sufficient magnitude to affect the overall treatment period interval (gestation days 6-20) in this group, and therefore was not considered test substance-related. Mean body weights in the 500 mg/kg/day group were significantly (p<0.05) lower than the control group during gestation days 18-20; given the small magnitude of change (only 3.8% to 4.0% lower compared to the control group), the lower mean body weights noted at this dosage level were not considered test substance-related. A significantly (p<0.05) lower mean net body weight change was noted at 500 mg/kg/day compared to the control group; however, mean net body weight in this group was similar to the control group, and therefore this change was not considered test substance-related.
Mean maternal body weights, net body weight, and net body weight gain in the 375 mg/kg/day group and gravid uterine weights in the 375 and 500 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, lower mean maternal food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 500 mg/kg/day group during gestation days 6-9 and in the 750 mg/kg/day group during gestation days 6-9 and 15-20 compared to the control group and resulted in lower mean food consumption in these groups when the entire treatment period (gestation days 6-20) was evaluated; differences were generally significant (p<0.05 or p<0.01). The lower mean food consumption in the 750 mg/kg/day group correlated with the lower mean body weights and body weight gains and was considered adverse. The differences noted in the 500 mg/kg/day group were generally not of sufficient magnitude to affect mean body weights and body weight gains in this group, and therefore were considered nonadverse. Mean food consumption at 500 and 750 mg/kg/day was similar to the control group during gestation days 9-12 and 12-15.
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 375 mg/kg/day group was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05) lower mean g/animal/day food consumption values were noted in the 375 mg/kg/day group compared to the control group during gestation days 6-9 and 6-20; however, these differences were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were not considered test substance-related.
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
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 test substance-related effects on mean lung weights were noted in the 375, 500, and 750 mg/kg/day groups. Differences from the control group were slight and not statistically significant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 375, 500, and 750 mg/kg/day. Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related. With the exception of 2 and 1 females in the control and 750 mg/kg/day groups, respectively, all females were determined to be gravid.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
With the exception of 2 and 1 females in the control and 750 mg/kg/day groups, respectively, all females were determined to be gravid.
Other effects:
not specified
Details on maternal toxic effects:
Additional parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male, female, and combined fetal weights in the 750 mg/kg/day group were lower (5.1%, 7.9%, and 5.3%, respectively) than the control group; differences were significant (p<0.01) and were considered test substance-related. The lower mean fetal weights at 750 mg/kg/day were attributed to the decrements in mean body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth. Mean male, female, and combined fetal weights at 375 and 500 mg/kg/day and survival at all dosage levels were unaffected by test substance administration.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean male, female, and combined fetal weights at 375 and 500 mg/kg/day were unaffected by test substance administration. The lower mean fetal weights at 750 mg/kg/day were attributed to the decrements in mean maternal body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Live litter sizes were similar across all groups. Mean male, female, and combined fetal weights at 375 and 500 mg/kg/day were unaffected by test substance administration. The lower mean fetal weights at 750 mg/kg/day were attributed to the decrements in mean maternal body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Fetal survival at all dosage levels was unaffected by test substance administration.
External malformations:
no effects observed
Description (incidence and severity):
No external malformations or developmental variations were observed in fetuses in this study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of fetuses (litters) with skeletal malformations were 1(1), 2(2), and 3(3), and 0(0) in the control, 375, 500, and 750 mg/kg/day groups, respectively. A vertebral anomaly with an associated rib anomaly (fused, misshapen, and/or malpositioned arches; fused malpositioned, absent, and/or malproportioned centra; extra costal cartilage; fused ribs; moderately malaligned sternebrae; 12 left ribs and 11 right ribs present; 25 presacral vertebrae) was noted for 1 fetus each in the 375 (no. 41462-08) and 500 (no. 41525-05) mg/kg/day groups. In the 500 mg/kg/day group, fetus no. 41431-10 was noted with a bent scapula (right) and fetus no. 41459-05 was noted with a costal cartilage anomaly (fused costal cartilage); 1 fetus in the control group (no. 41466-09) was also noted with a costal cartilage anomaly. A rib anomaly (fused and misshapen arches; fused and malpositioned centra; fused ribs; extra costal cartilage; 12 left ribs and 11 right ribs present; moderately malaligned sternebrae; 25 presacral vertebrae) was noted for fetus no. 41498-15 in the 375 mg/kg/day group. The aforementioned skeletal malformations were observed in single fetuses, noted similarly in the control group, did not occur in a doserelated manner, and/or the mean litter proportions were within Charles River Ashland historical control data ranges; therefore, they were not considered test substance-related.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral malformations were limited to 1 fetus (no. 41522-13) in the 500 mg/kg/day group noted with hydrocephaly (increased cavitation of both the lateral and third ventricles). Because this finding was noted in a single fetus, did not occur in a dose-related manner, and the value was within the range of the Charles River Ashland historical control data, it was not considered test substance-related.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 2 (nos. 41454-04 and 41466-06), 4 (nos. 41479-06, 41479-11, 41479-14, and 41519-09), and 1 (no. 41502-06) fetuses in the control, 500, and 750 mg/kg/day groups, respectively. Fetus no. 41472-12 in the 500 mg/kg/day group was noted with a white area on the liver. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 305(22), 329(23), 328(24), and 299(23) in the control, 375, 500, and 750 mg/kg/day groups, respectively. Malformations were observed in 1(1), 2(2), 4(4), and 0(0) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
Treatment related:
yes

The analyzed dosing formulations were within Charles River SOP range for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

Results of the analyses of dosing formulations are summarized below.

Table 7. Results of Homogeneity and Concentration Analyses

  Group 2 (75 mg/mL) Group 3 (100 mg/mL) Group 4 (150 mg/mL)
Homogeneity and Concentration Assessment of the 08 Jun 2016 Formulations
Mean Concentration (mg/mL) 78.3 106 156
RSD (%) 0.15 0.22 0.46
Mean % of Target 104 106 104
9-Day Refrigerated Storage Resuspension Homogeneity Assessment of the 08 Jun 2016 Formulations
Mean Concentration (mg/mL) 75.8 NA 153
RSD (%) 0.77 NA 0.088
Mean % of Target 101 NA 102
Homogeneity and Concentration Assessment of the 24 Jun 2016 Formulations
Mean Concentration (mg/mL) 79.7 106 155
RSD (%) 0.48 0.21 0.28
Mean % of Target 106 106 103
Conclusions:
Adverse mean body weight losses and lower mean body weight gains with corresponding reduced mean food consumption were noted in the 750 mg/kg/day group, with the greatest magnitude of change occurring during the fetal period, which resulted in lower mean body weights, net body weight, net body weight gain, and gravid uterine weight in this group. The aforementioned decrements in body weight and food consumption parameters at 750 mg/kg/day noted during the period of greatest fetal growth (gestation days 15-20) resulted in lower mean fetal weights in this group. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when n-butyl pyrrolidone was administered by dermal application to bred Crl:CD(SD) rats.
Executive summary:

The objectives of the study were to determine the potential of the test substance, N-butylpyrrolidone, to induce developmental toxicity after maternal dermal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. The test substance, N-butyl pyrrolidone, in the vehicle (deionized water) was administered by dermal application to the dorsal scapular area (approximately 10% of total body surface) to 3 groups of bred female Crl:CD(SD) rats once daily from gestation days 6 through 19; animals were fitted with Elizabethan collars and exposed to the test substance for approximately 23 hours each day. Exposure levels were 375, 500, and 750 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. In the original study design, each group consisted of 25 bred females. However, the 23 females that were dosed on 13 Jun 2016 (gestation day 6) did not have proper access to food because they were provided the incorrect food jars for use with Elizabethan collars. This resulted in severe body weight loss and reduced food consumption for these females from gestation day 6 to 7. Therefore, these females were removed from study on 14 Jun 2016 and 18 additional females were assigned to the study. Based on the number of animals available for replacement, the total number of pregnant females assigned to the control, 375, 500, and 750 mg/kg/day groups was 24, 23, 24, and 24, respectively.

All animals were observed twice daily for mortality and moribundity. Clinical and dermal observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female and the lungs were weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

Results

All females in the control, 375, 500, and 750 mg/kg/day groups survived to the scheduled necropsy. A slightly increased incidence of red material around the eyes was noted in all test substance-treated groups generally throughout the treatment period at the daily examinations; however, given the low occurrence of this finding (generally 1-4 occurrences per animal) and the inability of the animals to groom while outfitted with the Elizabethan collars, this finding was considered nonadverse. No other test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration in the 375, 500, and 750 mg/kg/day groups. No dermal findings were noted at any dosage level.

Lower mean body weight gains were noted in the 375, 500, and 750 mg/kg/day groups compared to the control group prior to the initiation of dose administration (gestation days 0-6); mean body weights in these groups were similar to the control group during this interval. Mean body weight losses were noted in all groups, including the control group, following the initiation of dose administration (gestation day 6-7); these differences were likely due to the placement of the Elizabethan collars. Test substance-related mean body weight losses or lower mean body weight gains were noted in the 750 mg/kg/day group compared to the control group during gestation days 6-9 and 15-20, and resulted in a lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated; mean body weight gains in this group were similar to the control group during gestation days 9-12 and 12-15. In addition, mean body weights in the 750 mg/kg/day group were up to 7.4% lower than the control group during gestation days 8-20. A test substance-related lower mean net body weight and net body weight gain were also noted in the 750 mg/kg/day group. The aforementioned body weight effects noted in the 750 mg/kg/day group were considered adverse. The mean gravid uterine weight in the 750 mg/kg/day group was lower than the control group and was considered test substance-related. Mean body weight gains in the 375 and 500 mg/kg/day groups were generally similar to the control group for the remainder of the treatment period (gestation days 6-9, 9-12, 12-15, 15-20, and 6-20). Mean body weights in the 500 mg/kg/day group were up to 4.0% lower than the control group during gestation days 18-20; given the small magnitude of change and absence of a corresponding effect on mean body weight gain, the lower mean body weights noted at this dosage level were not considered test substance-related. A lower mean net body weight change was noted at 500 mg/kg/day compared to the control group; however, mean net body weight in this group was similar to the control group, and therefore this change was not considered test substance-related. Test substance-related, lower mean maternal food consumption was noted in the 500 and 750 mg/kg/day groups during gestation days 6-9 and/or 15-20 compared to the control group and resulted in lower mean food consumption in these groups when the entire treatment period (gestation days 6-20) was evaluated; mean food consumption in these groups was similar to the control group during gestation days 9-12 and 12-15. The lower mean food consumption in the 750 mg/kg/day group correlated with the lower mean body weights and body weight gains and was considered adverse; the differences noted in the 500 mg/kg/day group were generally not of sufficient magnitude to affect mean body weights and body weight gains, and therefore were considered nonadverse. Mean maternal body weights, net body weight, net body weight gain, and food consumption in the 375 mg/kg/day group and gravid uterine weights in the 375 and 500 mg/kg/day groups were unaffected by test substance administration.

No test substance-related macroscopic findings or changes in mean lung weights were noted for females at any dosage level at the scheduled necropsy on gestation day 20.

Mean male, female, and combined fetal weights in the 750 mg/kg/day group were 5.1%, 7.9%, and 5.3% lower, respectively, than the control group; the differences were considered test substance-related and attributed to the decrements in mean body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth. Intrauterine growth at 375 and 500 mg/kg/day and survival at 375, 500, and 750 mg/kg/day were unaffected by test substance administration. There were no test substance-related external, visceral, and skeletal malformations or developmental variations noted at any dosage level.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-11 to 2016-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The actual ESR with UUID b01c04cd-9699-4682-ae06-f1c56743d4ad has been deleted as recommended by ECHA (INC 198666). Due to a false positive virus alert, the dossier got rejected several times. The original content of the study (including the formerly attached Summary Tables) has been reinstalled in a new RSS.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at room temperature under a nitrogen blanket, and was considered stable under these conditions.
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Crl:CD(SD) rats (155 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 26-Jan-2016.
- Age at study initiation: 79 days old upon receipt; 13 weeks old when paired for breeding.
- Weight at study initiation: Body weight values ranged from 220 g to 296 g on gestation day 0.
- Fasting period before study: no
- Housing: Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- Diet (e.g. ad libitum): ad libitum. The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Water (e.g. ad libitum): ad libitum. Reverse osmosis-purified (on-site) drinking water, delivered byan automatic watering system.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71°F ± 5°F (22°C ± 3°C). Actual mean daily temperature ranged from 70.6°F to 70.8°F (21.4°C to 21.6°C) during the study.
- Humidity (%): 50% ± 20%. Actual mean daily relative humidity ranged from 44.1% to 53.5% during the study.
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 26-Jan-2016 Experimental starting date (animal receipt)
To: 03-Mar-2016 (Last laparohysterectomy)
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated (2°C to 8°C), purged with nitrogen. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
Dosing formulations were prepared at the test substance concentrations indicated in the table (see below).
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2°C to 8°C), purged with nitrogen. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
The first test substance dosing formulations were visually inspected by the Study Director’s designee and were found to be visibly homogeneous and acceptable for administration.

VEHICLE
- Concentration in vehicle: 0, 40, 60, 80 and 100 mg/mL for the dosage levels of 0, 200, 300, 400 and 500 mg/kg bw/day, respectively.
- Amount of vehicle (if gavage): The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.
- Purity: The vehicle used in preparation of the test substance formulations and for administration to the control group was deionized water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance in the vehicle was previously established to be stable and homogenous following 11 days of refrigerated and room temperature storage at concentrations of 40 and 100 mg/mL. Samples for homogeneity and/or concentration determination were collected from the top, middle, an d bottom strata of the first 40 and 100 mg/mL dosing formulations and from the middle stratum of the first control, 60, and 80 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determination were collected from the top and bottom strata of an aliquot taken from the first 40 and 100 mg/mL dosing suspensions following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum of the last dosing formulations (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored refrigerated (2°C to 8°C) and protected from light as back-ups, and the first dosing formulations were also stored purged with nitrogen. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: not specified.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
single exposure
Frequency of treatment:
once daily
Duration of test:
during gestation days 6-19
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Details on study design:
- Dose selection rationale: Dosage levels were determined from results of previous studies (Hansen, 2013; see disregarded study LPT, 2013) and were discussed by the Sponsor Representative and the Study Director.
- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research colony.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20. When body weights could not be determined for an animal during a given in terval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were designated as “NA” on the individual report tables.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

POST-MORTEM EXAMINATIONS: Yes
A gross necropsy was performed on the female that was found dead during the course of the study. The number and location of implantation sites and corpora lutea were recorded. The female and all products of conception were discarded.
- Sacrifice on gestation day 20.
- Organs examined: Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving rats were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded.

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% am monium sulfide solution for detection of early implantation loss (Salewski, 1964).
Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded (see Deviations from the Protocol). Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). Fetuses were then examined for skeletal malformations and developmental variations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Statistics:
please see below ("Any other information on materials and methods incl. tables").
Indices:
Gestation Day 20 Laparohysterectomy
Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group)/ No. Gravid Females/Group
2. Proportional Litter Basis:
Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group;
Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter)/ No. Implantation Sites/Litter x 100
Fetal Morphological Examination
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%))/ No. Litters/Group
Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter)/ No. Viable Fetuses/Litter x 100
Historical control data:
WIL Research has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related clinical observations were noted in the 500 mg/kg/day group and consisted of rales and clear and red material around the mouth. Rales were noted in the 500 mg/kg/day group at the daily examinations and approximately 1 hour following dose administration generally throughout the treatment period (gestation days 7-20) and were considered adverse. Clear and red material around the mouth were noted for females in the 500 mg/kg/day group approximately 1 hour following dose administration. Because the material findings were noted primarily once per animal, they were not considered adverse. Rales were also noted in the 300 and 400 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration. However, due to the limited incidence of this finding in these groups, rales were considered test substance-related but not adverse. No other test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Findings noted in the treated groups, including hair loss on the forelimbs, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female no. 9857 in the 500 mg/kg/day group was found dead on gestation day 10. No remarkable clinical observations or effects on body weight or food consumption were noted for this female prior to death and the cause of death was not determined at necropsy. Therefore, the death in the 500 mg/kg/day group was not attributed to test substance administration. All other animals survived to the scheduled necropsy on gestation day 20.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related significant (p<0.01) mean body weight loss was noted in the 500 mg/kg/day group compared to a body weight gain in the control group during gestation days 6-9 due to mean body weight losses noted in this group immediately following the initiation of dose administration (gestation day 6-8, significant [p<0.01] during gestation days 6-7 only). Mean body weight gain in this group was comparable to the control group during the remainder of the treatment period (gestation days 9-12, 12-15 and 15-20). As a result of the initial mean body weight loss noted in the 500 mg/kg/day group, a significantly (p<0.05) lower mean body weight gain was observed for this group compared to the control group when the overall treatment period (gestation days 6-20) was evaluated. In addition, mean body weights in the 500 mg/kg/day group were 5.0% to 6.0% lower than the control group during gestation days 9-15; the differences were significant (p<0.05 or p<0.01). Mean net body weight gain in the 500 mg/kg/day group was also significantly (p<0.01) lower than the control group. Mean net body weight and gravid uterine weight in this group were comparable to the control group.
In the 200, 300, and 400 mg/kg/day groups, test substance-related lower mean body weight gains were noted during gestation days 6-9 compared to the control group due to mean body weight losses noted in these groups immediately following the first dose (gestation day 6-7); the difference was significant (p<0.05) for the 400 mg/kg/day group. Mean body weight gains in these groups were comparable to the control group throughout the remainder of the treatment period (gestation days 9-12, 12-15, and 15-20). Because the initial mean body weight losses observed in the 200, 300, and 400 mg/kg/day groups did not affect the overall mean body weight gain (gestation days 6-20) or mean body weights in these groups, they were not considered adverse. Mean net body weight gains in the 200, 300, and 400 mg/kg/day groups were lower than the control group; the differences at 300 and 400 mg/kg/day were significant (p<0.05 or p<0.01). Mean net body weights and gravid uterine weights in these groups were comparable to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 500 mg/kg/day group during gestation days 6-9 and 9-12; the differences were significant (p<0.01) compared to the control group and corresponded to lower mean body weight gains. Mean food consumption in this group was comparable to the control group during gestation days 12-15 and 15-20. As a result of the lower mean food consumption noted in the 500 mg/kg/day group during the first week of the treatment period, mean food consumption in this group was significantly (p<0.01) lower than the control group when the overall treatment period (gestation days 6-20) was evaluated.
In the 200, 300, and 400 mg/kg/day groups, significantly (p<0.01) lower mean food consumption corresponding to mean body weight losses was noted immediately following the first dose (gestation day 6-7). However, these differences were transient and did not affect the overall treatment period (gestation days 6-20) and therefore were not considered adverse. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Female no. 9857 in the 500 mg/kg/day group was found dead on gestation day 10. This female had 17 normally developing implantations in utero and no macroscopic findings were noted at necropsy. At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 200, 300, 400, and 500 mg/kg/day. Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose related.
One female each in the control and 200 mg/kg/day groups were nongravid.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
Intrauterine growth in the 200 mg/kg/day group and survival in the 200, 300, 400, and 500 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The mean litter proportions of pre-implantation loss were similar across all groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female each in the control and 200 mg/kg/day groups were nongravid.
Other effects:
no effects observed
Details on maternal toxic effects:
Developmental toxicity was manifested at a dosage level of 500 mg/kg/day. Test substance-related statistically significantly lower (up to 10.0%) mean male, female, and combined fetal body weights were noted in the 500 mg/kg/day group compared to the concurrent control group. There were no test substance-related effects on intrauterine survival or fetal morphology at 500 mg/kg/day. The developmental toxicity noted at 500 mg/kg/day occurred in the presence of maternal toxicity.
At the daily examinations and approximately 1 hour following dose administration, test substancerelated higher incidences of rales were observed at 500 mg/kg/day. In addition, clear and red material around the mouth were noted for females in the 500 mg/kg/day group approximately 1 hour following dose administration. Although these material findings were considered test substance-related, they were not considered adverse because these findings were noted primarily once per animal. A test substance-related mean body weight loss was noted in the 500 mg/kg/day group during gestation days 6-9 which resulted in a lower mean body weight gain in this group when the overall treatment period (gestation days 6-20) was evaluated and mean body weights that were 5.0% to 6.0% lower than the control group during gestation days 9-15. In addition, lower mean food consumption was noted in the 500 mg/kg/day group compared to the control group during gestation days 6-9 and 9 -12 and when the overall treatment period (gestation days 6-20) was evaluated. In the 300 and 400 mg/kg/day groups, rales were noted sporadically throughout the treatment period and therefore were considered test substance-related, but not adverse. Test substance-related lower mean body weight gains and corresponding lower mean food consumption were noted for the 200, 300, and 400 mg/kg/day groups during gestation days 6-9 due to mean body weight losses and lower mean food consumption noted in these groups immediately following the first dose (gestation day 6-7). In the absence of effects on mean body weight gains and food consumption when the overall treatment period was evaluated or mean body weights throughout the study, these transient differences were not considered to be adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male, female, and combined fetal body weights in the 500 mg/kg/day group (3.7 g, 3.6 g, and 3.6 g, respectively) were 7.7% to 10.0% lower than the concurrent control group (4.1 g, 3.9 g, and 4.0 g, respectively) and the values were lower than the mean values in the WIL Research historical control data ranges (3.898 g, 3.697 g, and 3.801 g for male, female, and combined, respectively). The differences in fetal body weights observed in the 500 mg/kg/day group were significant (p<0.01) compared to the concurrent control group (even when the difference in mean number of viable fetuses was incorporated) and considered test substance-related. Significantly (p<0.05 or p<0.01) lower mean fetal body weights (male, female, and combined) were also noted in the 300 and 400 mg/kg/day groups compared to the concurrent control group. The mean fetal body weight values in the 300 mg/kg/day and 400 mg/kg/day groups (3.9 g, 3.7 g, and 3.8 g for male, female, and combined, respectively, for both groups) were comparable to the WIL Research historical control mean values for these parameters (3.9 g, 3.7 g, and 3.8 g, for male, female, and combined, respectively). In addition, the concurrent control group mean values for these parameters (4.1 g, 3.9 g, and 4.0 g for male, female, and combined, respectively) were at the maximum mean values in the WIL Research historical control database comprised of 80 data sets. Furthermore, the magnitude of the differences in fetal weights between the 300 and 400 mg/kg/day groups and the concurrent control group was small (4.9% to 5.1% lower than the concurrent control group). Therefore, the differences in fetal body weights observed in the 300 and 400 mg/kg/day groups were considered to be incidental due to the lower number of fetuses and increased fetal body weights in the concurrent control group and not related to test substance administration.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of viable fetuses in the control group was 4% to 8% less than the test substance treated groups, although this difference was not statistically significant. The mean number of viable fetuses in the control group (13.4 fetuses) was at the lower end of the WIL Research historical control data range of control group values (mean = 14.63, minimum = 12.35, maximum = 16.82, 25th quartile = 14.24, and 75th quartile = 15.01).
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological evaluation were 322(24), 348(24), 352(25), 348(25), and 347(24) in the control, 200, 300, 400, and 500 mg/kg/day groups, respectively. Malformations were observed in 1(1), 1(1), 1(1), 2(2), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. External malformations were observed in 0(0), 1(1), 1(1), 0(0), and 1(1) fetuses (litters) in the control, 200, 300, 400, and 500 mg/kg/day groups, respectively. Fetus no. 9825-18 in the 200 mg/kg/day group and fetus no. 9766-16 in the 500 mg/kg/day group were observed with microphthalmia and anophthalmia, respectively. Skeletally these malformations consisted of orbits smaller than normal.
In the 300 mg/kg/day group, fetus no. 9851-07 had mandibular micrognathia that consisted of small mandibles that were fused along the mandibular symphysis. The external malformations observed in the 200, 300, and 500 mg/kg/day groups were noted in single fetuses, did not occur in a dose-related manner, and/or the mean litter proportions of these findings were not statistically significantly different from the concurrent control group and therefore they were not attributed to the test substance.
There were no external developmental variations noted for fetuses in this study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were observed in 1(1), 0(0), 0(0), 2(2), and 1(1) fetuses (litters) in the control, 200, 300, 400, and 500 mg/kg/day groups, respectively. One fetus each in the control, 400, and 500 mg/kg/day groups had sternoschisis (sternal band nos. 2 and/or 3 not joined). In addition, fetus no.9738-10 in the 400 mg/kg/day group had bent limb bones (femurs, fibula, tibia, humeri, radii, and ulnae). Because the skeletal malformations were noted for single fetuses, were observed similarly in the concurrent control group, and the mean litter proportions were not statistically significantly different from the concurrent control group and/or were within the WIL Research historical control data, they were not considered to be test substance-related.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no visceral malformations noted for fetuses in this study.
No test substance-related visceral developmental variations were noted at any dosage level. Findings observed in the test substance-treated groups were noted infrequently, similarly in the concurrent control group, and/or were not observed in a dose-related manner. Furthermore, the differences in the mean litter proportions of these findings were not statistically significant compared to the concurrent control group and/or the values were within the ranges of the WIL Research historical control data. Red discoloration of the eye was observed in fetus no. 9818-08 in the 500 mg/kg/day group and renal papilla(e) not fully developed (Woo and Hoar grade 1) were noted for fetus nos. 9788-01 and 9834-03 in the control and 500 mg/kg/day groups, respectively. These findings were not classified as either malformations or developmental variations, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Other effects:
no effects observed
Description (incidence and severity):
Developmental toxicity was manifested at a dosage level of 500 mg/kg/day. Test substance-related statistically significantly lower (up to 10.0%) mean male, female, and combined fetal body weights were noted in the 500 mg/kg/day group compared to the concurrent control group. There were no test substance-related effects on intrauterine survival or fetal morphology at 500 mg/kg/day. The developmental toxicity noted at 500 mg/kg/day occurred in the presence of maternal toxicity.
Although mean fetal body weights in the 300 and 400 mg/kg/day groups were statistically significantly lower (approximately 5%) than the concurrent control group, the values in these groups were equal to the mean fetal body weight values in the WIL Research historical control database (comprised 80 data sets). In addition, the mean fetal body weight values within the concurrent control group were the maximum values found within the WIL Research historical control database. The very high mean fetal body weight values within the concurrent control group may be due to the lower number of mean viable fetuses within this group. Therefore, the differences in mean fetal body weight in the 300 and 400 mg/kg/day groups were considered incidental due to the lower number of viable fetuses and increased fetal body weights in the concurrent control group and not related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
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:
not specified

Analyses of Dosing Formulations

The analyzed dosing formulations were within WIL Research SOP range for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).

Results of the analyses of dosing formulations are summarized below.

Text Table 1. Results of Homogeneity Analyses
  Group 2
(40 mg/mL)
Group 5
(100 mg/mL)
Homogeneity Assessment of the 12-Feb-2016 Formulations
Mean Concentration (mg/mL) 40.0 103
RSD (%) 0.57 0.57
Mean % of Target 100 103
10-Day Room Temperature Resuspension Homogeneity Assessment of the 12-Feb-2016 Formulations
Mean Concentration (mg/mL) 40.5 102
RSD (%) 0.53 0.17
Mean % of Target 101 102

Text Table 2. Results of Concentration Analyses
  Mean Concentration, mg/mL (% of Target)
  Group 2     Group 3 Group 4 Group 5
Date of Preparation (40 mg/mL) (60 mg/mL) (80 mg/mL) (100 mg/mL)
12-Feb-2016 40.0 (100)  61.3 (102) 81.2 (102) 103 (103)
25-Feb-2016 40.6 (102)  61.8 (103) 82.5 (103) 102(102)

The summary tables (S1 - S15) depicted below show the results of the evaluated parameters.

Table S1

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of maternal survival and pregnancy status

Dose group :

1

2

3

4

5

No.

%

No.

%

No.

%

No.

%

No.

%

Females on study

25

25

25

25

25

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

Females that died

0

0.0

0

0.0

0

0.0

0

0.0

1

4.0

Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

nongravid

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

gravid

0

0.0

0

0.0

0

0.0

0

0.0

1

100.0

Females that were euthanized

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

nongravid

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

gravid

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

Females examined at scheduled necropsy

25

100.0

25

100.0

25

100.0

25

100.0

24

96.0

nongravid

1

4.0

1

4.0

0

0.0

0

0.0

0

0.0

gravid

24

96.0

24

96.0

25

100.0

25

100.0

24

100.0

with resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

0

0.0

with viable fetuses

24

100.0

24

100.0

25

100.0

25

100.0

24

100.0

Total females gravid

24

96.0

24

96.0

25

100.0

25

100.0

25

100.0

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

Table S2  (daily examinations)

 

 

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of clinical findings: total occurrence/no. of animals

----- f e m a l e -----

Table range:

02 -09 -16 to 03 -03-16

Group:

1

2

3

4

5

normal

-no significant clinical observations

489/25

508/25

505/25

472/25

451/24

disposition

-found dead

0/0

0/0

0/0

0/0

1/1

-scheduled euthanasia; gestation day 20

25/25

25/25

25/25

25/25

24/24

body/integument

-hair loss forelimb(s)

32/ 4

14/2

7/ 2

50/4

44/ 4

-hair loss ventral trunk

0/0

2/1

10/1

0/0

0/0

-hair loss facial area

3/ 1

0/0

0/0

0/0

0/0

cardio-pulmonary

-rales

0/0

0/0

0/0

3/ 3

28/10

eyes/ears/nose

-dried red material around nose

0/0

1/1

1/1

0/0

1/1

body/integ. II

-wet yellow material urogenital area

1/1

2/ 2

2/1

0/0

0/0

-wet yellow material ventral trunk

1/1

0/0

0/0

0/0

0/0

-dried brown material facial area

0/0

1/1

0/0

0/0

0/0

body/integ. II

-dried yellow material dorsal head

0/0

1/1

0/0

0/0

0/0

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

Table S3

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of post-dose findings: total occurrence/no. of animals

----- f e m a l e -----

Table range:

02-15-16 to 03-02-16

Group:

1

2

3

4

5

normal

1 hour post-dosing

-no significant clinical observations

350/25

348/25

347/25

313/25

294/25

unsched obs (>7s mins)

-no significant clinical observations

0/0

0/0

0/0

24/24

0/0

cardio-pulmonary

1 hour post-dosing

-rales

0/0

0/0

2/2

8/6

28/12

eyes/ears/nose

1 hour post-dosing

-dried red material around nose

0/0

0/0

0/0

0/0

3/3

oral/dental

1 hour post-dosing

-wet clear material around mouth

0/0

1/1

0/0

2/2

8/5

-dried red material around mouth

0/0

1/1

1/1

3/3

6/6

-dried clear material around mouth

0/0

0/0

0/0

0/0

3/3

special ii

time of dose

-undetermined amount of dose expelled

0/0

0/0

0/0

1/1

1/1

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

Table S4

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of body weights during gestation [g]

group:

0 mg/kg/day

200 mg/kg/day

300 mg/kg/day

400 mg/kg/day

500 mg/kg/day

Day 0         

mean

255.

255.

254.

255.

256.

% difference

0.0

-0.4

0.0

0.4

S.D.

13.0

15.4

13.6

15.0

15.7

S.E.

2.6

3.1

2.7

3.0

3.1

n

24

24

25

25

25

Day 6         

mean

287.

284.

283.

284.

283.

% difference

-1.0

-1.4

-1.0

-1.4

S.D.

15.5

17.6

14.3

16.3

17.6

S.E.

3.2

3.6

2.9

3.3

3.5

n

24

24

25

25

25

Day 7         

mean

289.

283.

281.

282.

279.

% difference

-2.1

-2.8

-2.4

-3.5

S.D.

15.0

17.5

14.3

15.9

18.5

S.E.

3.1

3.6

2.9

3.2

3.7

n

24

24

25

25

25

Day 8         

mean

292.

287.

284.

284.

279.

% difference

-1.7

-2.7

-2.7

-4.5

S.D.

16.9

15.8

14.1

15.4

17.9

S.E.

3.5

3.2

2.8

3.1

3.6

n

24

24

25

25

25

Day 9         

mean

297.

290.

288.

289.

281.**

% difference

-2.4

-3.0

-2.7

-5.4

S.D.

16.4

16.4

15.1

15.5

19.6

S.E.

3.3

3.4

3.0

3.1

3.9

n

24

24

25

25

25

Day 10         

mean

302.

297.

294.

294.

284.**

% difference

-1.7

-2.6

-2.6

-6.0

S.D.

16.1

18.1

13.6

16.3

19.3

S.E.

3.3

3.7

2.7

3.3

3.9

n

24

24

25

25

24

Day 11         

mean

307.

302.

299.

299.

289.**

% difference

-1.6

-2.6

-2.6

-5.9

S.D.

16.5

18.3

14.8

17.0

19.0

S.E.

3.4

3.7

3.0

3.4

3.9

n

24

24

25

25

24

Day 12         

mean

311.

308.

303.

303.

295.**

% difference

-1.0

-2.6

-2.6

-5.1

S.D.

18.2

18.2

15.3

18.2

20.8

S.E.

3.7

3.7

3.1

3.6

4.2

n

24

24

25

25

24

Day 13         

mean

317.

313.

309.

309.

301.*

% difference

-1.3

-2.5

-2.5

-5.0

S.D.

20.0

19.9

15.7

19.2

20.6

S.E.

4.1

4.1

3.1

3.8

4.2

n

24

24

25

25

24

Day 14         

mean

324.

318.

314.

314.

305.**

% difference

-1.9

-3.1

-3.1

-5.9

S.D.

18.0

18.9

16.1

18.6

23.0

S.E.

3.7

3.9

3.2

3.7

4.7

n

24

24

25

25

24

Day 15         

mean

330.

325.

320.

322.

312.**

% difference

-1.5

-3.0

-2.4

-5.5

S.D.

19.0

19.3

17.3

19.8

24.1

S.E.

3.9

3.9

3.5

4.0

4.9

n

24

24

25

25

24

Day 16         

mean

340.

336.

331.

330.

323.

% difference

-1.2

-2.6

-2.9

-5.0

S.D.

20.1

20.3

18.0

22.8

25.0

S.E.

4.1

4.2

3.6

4.6

5.1

n

24

24

25

25

24

Day 17         

mean

353.

348.

345.

345.

337.

% difference

-1.4

-2.3

-2.3

-4.5

S.D.

22.7

22.2

19.1

22.6

26.3

S.E.

4.6

4.5

3.8

4.5

5.4

n

24

24

25

25

24

Day 18         

mean

369.

366.

362.

360.

353.

% difference

-0.8

-1.9

-2.4

-4.3

S.D.

22.5

22.8

20.7

24.2

27.9

S.E.

4.6

4.7

4.1

4.8

5.7

n

24

24

25

25

24

Day 19         

mean

385.

382.

376.

375.

366.

% difference

-0.8

-2.3

-2.6

-4.9

S.D.

25.1

22.6

20.4

25.2

28.5

S.E.

5.1

4.6

4.1

5.0

5.8

n

24

24

25

25

24

Day 20         

mean

398.

397.

392.

387.

381.

% difference

-0.3

-1.5

-2.8

-4.3

S.D.

27.5

24.8

22.7

28.3

32.6

S.E.

5.6

5.1

4.5

5.7

6.7

n

24

24

25

25

24

*   = significantly different from the control group at 0.05 using Dunnett's test

** = significantly different from the control group at 0.01 using Dunnett's test

nongravid weight(s) not included in calculation of mean

Table S5

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of body weight changes during gestation [g]

Group:

0 mg/kg/day

200 mg/kg/day

300 mg/kg/day

400 mg/kg/day

500 mg/kg/day

Day     0- 6

mean

33.

28.

28.

29.

27.

S.D.

6.5

5.3

6.7

7.1

6.5

S.E.

1.3

1.1

1.3

1.4

1.3

n

24

24

25

25

25

Day     6- 7

mean

2.

-1.

-2.*

-2.*

-3.**

S.D.

3.3

3.4

3.2

4.8

6.0

S.E.

0.7

0.7

0.6

1.0

1.2

n

24

24

25

25

25

Day     7- 8

mean

3.

5.

3.

2.

-1.

S.D.

4.9

5.3

4.2

5.4

7.1

S.E.

1.0

1.1

0.8

1.1

1.4

n

24

24

25

25

25

Day     8- 9

mean

5.

3.

4.

5.

2.

S.D.

5.7

5.0

4.3

5.8

6.0

S.E.

1.2

1.0

0.9

1.2

1.2

n

24

24

25

25

25

Day     9- 10

mean

5.

7.

5.

5.

4.

S.D.

3.8

5.3

5.8

3.7

6.5

S.E.

0.8

1.1

1.2

0.7

1.3

n

24

24

25

25

24

Day   10- 11

mean

5.

5.

5.

4.

5.

S.D.

4.5

3.9

4.7

4.2

5.6

S.E.

0.9

0.8

0.9

0.8

1.1

n

24

24

25

25

24

Day   11- 12

mean

4.

6.

4.

4.

5.

S.D.

5.0

4.8

6.9

5.3

5.6

S.E.

1.0

1.0

1.4

1.1

1.2

n

24

24

25

25

24

Day   12- 13

mean

6.

5.

6.

6.

6.

S.D.

6.2

6.7

6.8

5.3

5.3

S.E.

1.3

1.4

1.4

1.1

1.1

n

24

24

25

25

24

Day   13- 14

mean

7.

5.

5.

5.

4.

S.D.

5.7

5.7

5.9

5.7

8.1

S.E.

1.2

1.2

1.2

1.1

1.7

n

24

24

25

25

24

Day   14- 15

mean

6.

7.

7.

8.

7.

S.D.

5.2

4.4

3.7

3.8

6.2

S.E.

1.1

0.9

0.7

0.8

1.3

n

24

24

25

25

24

Day   15- 16

mean

10.

11.

10.

8.

11.

S.D.

4.4

6.2

5.3

8.2

5.8

S.E.

0.9

1.3

1.1

1.6

1.2

n

24

24

25

25

24

Day   16- 17

mean

14.

12.

14.

15.

14.

S.D.

6.6

8.5

4.5

5.7

5.8

S.E.

1.4

1.7

0.9

1.1

1.2

n

24

24

25

25

24

Day   17- 18

mean

15.

18.

17.

15.

16.

S.D.

5.7

8.3

6.1

5.1

5.3

S.E.

1.2

1.7

1.2

1.0

1.1

n

24

24

25

25

24

Day   18- 19

mean

16.

15.

15.

15.

13.

S.D.

6.0

4.0

4.1

4.3

5.3

S.E.

1.2

0.8

0.8

0.9

1.1

n

24

24

25

25

24

Day   19- 20

mean

14.

16.

15.

13.

15.

S.D.

5.2

4.5

4.7

7.0

7.3

S.E.

1.1

0.9

0.9

1.4

1.5

n

24

24

25

25

24

Day       6- 9

mean

10.

7.

6.

5.*

-2.**

S.D.

5.2

4.8

4.1

4.0

9.1

S.E.

1.1

1.0

0.8

0.8

1.8

n

24

24

25

25

25

Day     9- 12

mean

14.

17.

15.

14.

15.

S.D.

7.5

6.2

8.0

6.6

9.8

S.E.

1.5

1.3

1.6

1.3

2.0

n

24

24

25

25

24

Day   12- 15

mean

19.

18.

17.

19.

17.

S.D.

6.6

6.1

6.7

5.1

9.0

S.E.

1.4

1.2

1.3

1.0

1.8

n

24

24

25

25

24

Day   15- 20

mean

68.

72.

71.

65.

69.

S.D.

11.4

9.0

10.3

13.6

13.1

S.E.

2.3

1.8

2.1

2.7

2.7

n

24

24

25

25

24

Day     6- 20

mean

111.

113.

109.

103.

99.*

S.D.

16.2

12.2

14.5

19.2

22.9

S.E.

3.3

2.5

2.9

3.8

4.7

n

24

24

25

25

24

*   = significantly different from the control group at 0.05 using Dunnett's test

** = significantly different from the control group at 0.01 using Dunnett's test

mean differences calculated from individual differences

nongravid weight(s) not included in calculation of mean

Table S6

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of gravid uterine wts. and net body wt. changes [g]

Group:

0 mg/kg/day

200 mg/kg/day

300 mg/kg/day

400 mg/kg/day

500 mg/kg/day

initial body wt.

mean

255.

255.

254.

255.

255.

S.D.

13.0

15.4

13.6

15.0

15.8

S.E.

2.6

3.1

2.7

3.0

3.2

n

24

24

25

25

24

terminal body wt.

mean

398.

397.

392.

387.

381.

S.D.

27.5

24.8

22.7

28.3

32.6

S.E.

5.6

5.1

4.5

5.7

6.7

n

24

24

25

25

24

gravid uterine wt.

mean

81.0

84.6

82.9

79.9

80.0

S.D.

13.19

10.15

11.23

13.76

18.87

S.E.

2.69

2.07

2.25

2.75

3.85

n

24

24

25

25

24

net body wt.

mean

317.5

312.6

308.6

307.5

300.8

S.D.

18.87

19.06

19.25

21.19

19.87

S.E.

3.85

3.89

3.85

4.24

4.06

n

24

24

25

25

24

net body wt. change

mean

62.8

57.2

54.3*

52.3**

45.6**

S.D.

13.91

9.92

12.14

12.70

10.38

S.E.

2.84

2.02

2.43

2.54

2.12

n

24

24

25

25

24

*   = significantly different from the control group at 0.05 using Dunnett's test

** = significantly different from the control group at 0.01 using Dunnett's test

Table S7

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of food consumption during gestation [g/animal/day)

Group:

0 mg/kg/day

200 mg/kg/day

300 mg/kg/day

400 mg/kg/day

500 mg/kg/day

Day       0- 0

mean

20.

20.

20.

21.

20.

S.D.

2.0

2.0

1.7

1.6

1.8

S.E.

0.4

0.4

0.3

0.3

0.4

n                                                         

24

23

25

25

25

Day       6- 7

mean

21.

17.**

17.**

17.**

14.**

S.D.

2.4

2.0

2.7

4.7

3.4

S.E.

0.5

0.5

0.5

0.9

0.7

n

24

24

25

25

25

Day     7- 8

mean

22.

20.

18.**

21.

15.**

S.D.

2.9

2.7

2.9

2.8

4.5

S.E.

0.8

0.6

0.5

0.5

0.9

n

24

24

25

25

25

Day     8- 9

mean

22.

21.

21.

21.

17.**

S.D.

3.9

2.9

2.3

2.4

4'.5

S.E.

g.b

g.g

g.s

g.s

0.9

n

24

24

25

25

25

Day     9- 10

mean

23.

21.

21.

20.*

17.**

S.D.

2.7

3.5

3.3

3.5

4.2

S.E.

0.5

0.7

0.7

0.7

0.9

n

24

24

25

25

24

Day   10- 11

mean

22.

21.

21.

22.

19.**

S.D.

3.1

3.4

2.6

2.5

4.5

S.E.

0.6

0.7

0.5

0.5

0.9

n

24

24

25

25

24

Day   11- 12

mean

22.

21.

20.

21.

19.*

S.D.

3.2

3.1

3.6

2.8

4.2

S.E.

0.7

0.6

0.7

0.6

0.9

n

24

24

25

25

24

Day   12- 13

mean

22.

24.

24.

23.

21.

S.D.

4.4

4.8

4.8

3.7

3.7

S.E.

0.9

1.0

1.0

0.8

0.8

n

24

24

25

24

24

Day   13- 14

mean

23.

22.

22.

23.

20.

S.D.

4.5

5.3

5.7

5.2

5.3

S.E.

0.9

1.1

1.1

1.0

1.1

n

24

24

25

25

24

Day   14- 15

mean

23.

23.

22.

23.

22.

S.D.

3.5

3.2

5.3

3.0

4.3

S.E.

0.7

0.7

1.1

0.6

0.9

n

24

24

25

25

24

Day   15- 16

mean

24.

25.

24.

24.

22.

S.D.

2.7

3.8

2.6

3.1

3.5

S.E.

0.6

0.8

0.6

0.6

0.7

n

24

24

23

25

24

Day   16- 17

mean

26.

25.

26.

25.

24.

S.D.

3.1

3.5

2.4

3.7

3.3

S.E.

0.6

0.7

0.5

0.7

0.7

n

24

24

25

25

23

Day   17- 18

mean

25.

24.

24.

24.

23.

S.D.

4.6

3.1

4.1

2.9

4.2

S.E.

0.9

0.6

0.8

0.6

0.9

n

24

24

25

25

24

Day   18- 19

mean

26.

25.

25.

24.

24.

S.D.

4.7

4.3

2.9

3.0

4.9

S.E.

1.0

0.9

0.6

0.6

1.0

n

24

24

25

25

23

Day   19- 20

mean

21.

24.

22.

22.

22.

S.D.

6.9

2.8

5.2

5.4

3.0

S.E.

1.4

0.6

1.1

1.1

0.6

n

24

24

24

24

24

Day     6- 9

mean

21.

20.

19.**

20.

16.**

S.D.

2.7

1.8

1.8

2.0

3.3

S.E.

0.5

0.4

0.4

0.4

0.7

n

24

24

25

25

25

Day     9- 12

mean

22.

21.

21.*

21.

18.**

S.D.

2.3

2.3

1.7

2.1

3.5

S.E.

0.5

0.5

0.3

0.4

0.7

n

24

24

25

25

24

Day   12- 15

mean

23.

23.

23.

23.

21.

S.D.

2.5

3.1

3.8

2.8

3.5

S.E.

0.5

0.6

0.8

0.6

0.7

n

24

24

25

25

24

Day   15- 20

mean

24.

25.

24.

24.

23.

S.D.

2.9

2.7

2.2

2.5

2.6

S.E.

0.6

0.6

0.4

0.5

0.5

n

24

24

25

25

24

Day     6- 20

mean

23.

22.

22.

22.

20.**

S.D.

2.2

2.0

1.9

1.7

2.2

S.E.

0.5

0.4

0.4

0.3

0.5

n

24

24

25

25

24

*   = significantly different from the control group at 0.05 using Dunnett's test

** = significantly different from the control group at 0.01 using Dunnett's test

nongravid weight(s) not included in calculation of mean

Table S8

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of food consumption during gestation [g/kg/day]

Group:

0 mg/kg/day

200 mg/kg/day

300 mg/kg/day

400 mg/kg/day

500 mg/kg/day

Day     0- 6

mean

75.

73.

74.

77.

73.

S.D.

6.0

6.2

6.6

4.8

5.2

S.E.

1.2

1.3

1.3

1.0

1.0

n

24

23

25

25

25

Day     6- 7

mean

72.

61.**

59.**

60.**

50.**

S.D.

7.4

8.6

9.1

15.9

11.4

S.E.

1.5

1.8

1.8

3.2

2.3

n

24

24

25

25

25

Day     7- 8

mean

74.

70.

65.*

73.

55.**

S.D.

7.7

8.8

10.1

11.4

15.7

S.E.

1.6

1.8

2.0

2.3

3.1

n

24

24

25

25

25

Day     8- 9

mean

73.

74.

72.

75.

61.**

S.D.

11.3

10.0

7.1

9.3

14.6

S.E.

2.3

2.0

1.4

1.9

2.9

n

24

24

25

25

25

Day     9- 10

mean

77.

70.

71.

68.*

61.**

S.D.

8.5

10.8

11.9

10.4

13.7

S.E.

1.7

2.2

2.4

2.1

2.8

n

24

24

25

25

24

Day   10- 11

mean

73.

72.

70.

74.

65.*

S.D.

8.7

12.1

9.1

8.2

15.5

S.E.

1.8

2.5

1.8

1.6

3.2

n

24

24

25

25

24

Day   11- 12

mean

70.

70.

67.

71.

64.

S.D.

9.6

8.5

11.4

9.5

13.0

S.E.

2.0

1.7

2.3

1.9

2.7

n

24

24

25

25

24

Day   12- 13

mean

70.

77.

79.

75.

69.

S.D.

12.9

16.1

14.7

11.2

10.2

S.E.

2.6

3.3

2.9

2.3

2.1

n

24

24

25

24

24

Day   13- 14

mean

71.

70.

70.

73.

65.

S.D.

13.2

14.9

16.6

13.8

16.1

S.E.

2.7

3.0

3.3

2.8

3.3

n

24

24

25

25

24

Day   14- 15

mean

70.

72.

68.

72.

70.

S.D.

10.4

8.6

15.7

7.5

10.7

S.E.

2.1

1.8

3.1

1.5

2.2

n

24

24

25

25

24

Day   15- 16

mean

72.

75.

73.

74.

70.

S.D.

7.5

8.7

7.0

7.5

8.6

S.E.

1.5

1.8

1.5

1.5

1.7

n

24

24

23

25

24

Day   16- 17

mean

74.

71.

76.

74.

74.

S.D.

7.2

8.2

5.0

9.7

8.1

S.E.

1.5

1.7

1.0

1.9

1.7

n

24

24

25

25

23

Day   17- 18

mean

69.

68.

68.

69.

67.

S.D.

11.9

7.0

11.0

6.0

12.4

S.E.

2.4

1.4

2.2

1.2

2.5

n

24

24

25

25

24

Day   18- 19

mean

70.

67.

67.

67.

67.

S.D.

11.6

11.1

8.2

8.9

11.4

S.E.

2.4

2.3

1.6

1.8

2.4

n

24

24

25

25

23

Day   19- 20

mean

53.

61.

57.

57.

60.

S.D.

17.1

5.6

13.0

13.5

9.0

S.E.

3.5

1.2

2.7

2.8

1.8

n

24

24

24

24

24

Day     6- 9

mean

73.

68.

66.**

69.

55.**

S.D.

7.1

5.9

5.6

7.4

10.9

S.E.

1.4

1.2

1.1

1.5

2.2

n

24

24

25

25

25

Day     9- 12

mean

73.

70.

69.

72.

63.**

S.D.

6.3

6.8

5.7

6.5

11.2

S.E.

1.3

1.4

1.1

1.3

2.3

n

24

24

25

25

24

Day   12- 15

mean

70.

73.

72.

73.

68.

S.D.

6.6

8.9

10.7

6.0

8.3

S.E.

1.3

1.8

2.1

1.2

1.7

n

24

24

25

25

24

Day   15- 20

mean

67.

68.

68.

68.

67.

S.D.

6.8

5.4

5.6

5.2

6.4

S.E.

1.4

1.1

1.1

1.0

1.3

n

24

24

25

25

24

Day     6- 20

mean

70.

69.

68.

69.

64.**

S.D.

5.2

4.4

4.8

3.5

5.0

S.E.

1.1

0.9

1.0

0.7

1.0

n

24

24

25

25

24

*   = significantly different from the control group at 0.05 using Dunnett's test

** = significantly different from the control group at 0.01 using Dunnett's test

nongravid weight(s) not included in calculation of mean

Table S9

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of maternal macroscopic findings

Group:

1

2

3

4

5

number examined

25

25

25

25

25

no significant changes observed

23

23

23

25

23

nongravid -- ammonium sulfide negative

1

1

0

0

0

found dead

0

0

0

0

1

vagina: contents, brown

1

0

0

0

0

uterus: contents, dark red

0

0

0

0

1

spleen: area(s), white

0

1

0

0

0

placentae: pale

0

0

1

0

0

uterus: contents, brown fluid

0

0

1

0

0

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

Table S10

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of fetal data at scheduled necropsy

Group:

sex

viable fetuses

dead

resorptions

post implantation loss

implantation sites

corpora lutea

pre implantation loss

fetal weights in grams

no. of gravid females

m

f

fetuses

early

late

1

total

176

146

322

0

21

0

21

343

372

29

na

24

mean

7.3

6.1

13.4

0.0

0.9

0.0

0.9

14.3

15.5

1.2

4.0

S.D.

1.93

2.21

2.38

0.00

0.85

0.00

0.85

2.16

2.72

1.82

0.27

S.E.

0.39

0.45

0.48

0.00

0.17

0.00

0.17

0.44

0.55

0.37

0.05

2

total

160

188

348

0

22

1

23

371

387

16

na

24

mean

6.7

7.8

14.5

0.0

0.9

0.0

1.0

15.5

16.1

0.7

3.9

S.D.

1.97

1.71

1.93

0.00

0.88

0.20

0.91

1.72

2.25

1.27

0.26

S.E.

0.40

0.35

0.39

0.00

0.18

0.04

0.19

0.35

0.46

0.26

0.05

3

total

175

177

352

0

19

4

23

375

406

31

na

25

mean

7.0

7.1

14.1

0.0

0.8

0.2

0.9

15.0

16.2

1.2

3.8*

S.D.

2.16

1.98

2.18

0.00

0.97

0.80

1.41

1.83

2.28

1.90

0.21

S.E.

0.43

0.40

0.44

0.00

0.19

0.16

0.28

0.37

0.46

0.38

0.04

4

total

171

177

348

0

18

0

18

366

406

40

na

25

mean

6.8

7.1

13.9

0.0

0.7

0.0

0.7

14.6

16.2

1.6

3.8**

S.D.

1.93

1.98

2.48

0.00

0.79

0.00

0.79

2.55

2.30

2.68

0.22

S.E.

0.39

0.40

0.50

0.00

0.16

0.00

0.16

0.51

0.46

0.54

0.04

5

total

167

180

347

0

8

0

8

355

368

13

na

24

mean

7.0

7.5

14.5

0.0

0.3

0.0

0.3

14.8

15.3

0.5

3.6**

S.D.

2.44

2.55

3.44

0.00

0.56

0.00

0.56

3.49

3.67

0.78

0.20

S.E.

0.50

0.52

0.70

0.00

0.12

0.00

0.12

0.71

0.75

0.16

0.04

*   = significantly different from the control group at 0.05

** = significantly different from the control group at 0.01

na = not applicable

mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, fetal weights compared using dunnett's test

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

Table S11

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor: EASTMAN CHEMICAL Co.

Summary of fetal data at scheduled necropsy [% per litter]

Group:

0 mg/kg/day

200 mg/kg/day

300 mg/kg/day

400 mg/kg/day

500 mg/kg/day

corpora lutea

mean

15.5

16.1

16.2

16.2

15.3

S.D.

2.72

2.25

2.28

2.30

3.67

S.E.

0.55

0.46

0.46

0.46

0.75

n

24

24

25

25

24

implantation sites

mean

14.3

15.5

15.0

14.6

14.8

S.D.

2.16

1.72

1.83

2.55

3.49

S.E.

0.44

0.35

0.37

0.51

0.71

n

24

24

25

25

24

viable fetuses (%)

mean

93.7

93.8

93.9

95.2

97.8

S.D.

6.44

6.07

9.05

5.17

3.68

S.E.

1.32

1.24

1.81

1.03

0.75

n

24

24

25

25

24

dead fetuses (%)

mean

0.0

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

0.00

S.E.

0.00

0.00

0.00

0.00

0.00

n

24

24

25

25

24

early resorptions (%)

mean

6.3

6.0

5.0

4.8

2.2

S.D.

6.44

5.89

5.92

5.18

3.68

S.E.

1.32

1.20

1.18

1.04

0.75

n

24

24

25

25

24

late resorptions (%)

mean

0.0

0.3

1.1

0.0

0.0

S.D.

0.00

1.37

5.34

0.00

0.00

S.E.

0.00

0.28

1.07

0.00

0.00

n

24

24

25

25

24

total resorptions (%)

mean

6.3

6.3

6.1

4.8

2.2

S.D.

6.44

6.07

9.05

5.18

3.68

S.E.

1.32

1.24

1.81

1.04

0.75

n

24

24

25

25

24

pre-implantation loss (%)

mean

6.9

3.7

6.9

9.1

3.3

S.D.

9.48

5.89

9.96

14.43

4.69

S.E.

1.94

1.20

1.99

2.89

0.96

n

24

24

25

25

24

post-implantation loss (%)

mean

6.3

6.3

6.1

4.8

2.2

S.D.

6.44

6.07

9.05

5.18

3.68

S.E.

1.32

1.24

1.81

1.04

0.75

n

24

24

25

25

24

males (%)

mean

55.1

45.6

49.6

49.2

50.1

S.D.

13.13

10.93

13.09

13.26

15.80

S.E.

2.68

2.23

2.62

2.65

3.22

n

24

24

25

25

24

females (%)

mean

44.9

54.4

50.6

50.8

49.9

S.D.

13.13

10.93

13.09

13.26

15.80

S.E.

2.68

2.23

2.62

2.65

3.22

n

24

24

25

25

24

male fetal weights (g)

mean

4.1

3.9

3.9*

3.9**

3.7**

% difference

-4.9

-4.9

-4.9

 -9.8

S.D.

0.26

0.26

0.22

0.24

0.24

S.E.

0.05

0.05

0.04

0.05

0.05

n

24

24

25

25

24

female fetal weights (g)

mean

3.9

3.8

3.7*

3.7*

3.6**

% difference

-2.g

-5.1

-5.1

-7.7

S.D.

0.26

0.27

0.18

0.23

0.19

S.E.

0.05

0.06

0.04

0.05

0.04

n

24

24

25

25

23

combined fetal weights (g)

mean

4.0

3.9

3.8*

3.8**

3.6**

% difference

 - 2.5

 - 5.0

 - 5.0

 - 10.0

S.D.

0.27

0.26

0.21

0.22

0.20

S.E.

0.05

0.05

0.04

0.04

0.04

n

24

24

24

25

24

proportional (%) data compared using Dunn's test

corpora lutea and implantation sites compared using Dunnett's test

fetal weights compared using Dunnett's test

modified statistics used.

* indicates parametric analysis

 + indicates non-parametric analysis.

*   = significantly different from the control group at 0.05

** = significantly different from the control group at 0.01

Table S12

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor:EASTMAN CHEMICAL Co.

summary of fetuses and litters with malformations  [absolute no.]

Day 20

fetuses

litters

Dose group:

1

2

3

4

5

1

2

3

4

5

number examined externally

322

348

352

348

347

24

24

25

25

24

microphthalmia and/or anophthalmia

0

1

0

0

1

0

1

0

0

1

mandibular micrognathia

0

0

1

0

0

0

0

1

0

0

number examined viscerally

322

348

352

348

347

24

24

25

25

24

number with findings

0

0

0

0

0

0

0

0

0

0

number examined skeletally

322

348

352

348

347

24

24

25

25

24

sternoschisis

1

0

0

1

1

1

0

0

1

1

bent limb bone(s)

0

0

0

1

0

0

0

0

1

0

total number with malformations

external :

0

1

1

0

1

0

1

1

0

1

soft tissue :

0

0

0

0

0

0

0

0

0

0

skeletal :

1

0

0

2

1

1

0

0

2

1

combined :

1

1

1

2

1

1

1

1

2

1

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

Table S13

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor:EASTMAN CHEMICAL Co.

Summary of litter proportions of malformations % per litter   

day 20

Dose group:

1

2

3

4

5

number of litters examined externally

24

24

25

25

24

microphthalmia and/or anophthalmia

mean

0.0

0.2

0.0

0.0

0.3

S.D.

0.00

1.20

0.00

0.00

1.46

S.E.

0.00

0.25

0.00

0.00

0.30

mandibular micrognathia

mean

0.0

0.0

0.4

0.0

0.0

S.D.

0.00

0.00

2.00

0.00

0.00

S.E.

0.00

0.00

0.40

0.00

0.00

number of litters examined viscerally

24

24

25

25

24

number of litters with findings

0

0

0

0

0

number of litters examined skeletally

24

24

25

25

24

sternoschisis

mean

0.3

0.0

0.0

0.3

0.3

S.D.

1.28

0.00

0.00

1.54

1.46

S.E.

0.26

0.00

0.00

0.31

0.30

bent limb bone(s)

mean

0.0

0.0

0.0

0.4

0.0

S.D.

0.00

0.00

0.00

2.22

0.00

S.E.

0.00

0.00

0.00

0.44

0.00

total malformations

percent per litter with external malformations

mean

0.0

0.2

0.4

0.0

0.3

S.D.

0.00

1.20

2.00

0.00

1.46

S.E.

0.00

0.25

0.40

0.00

0.30

percent per litter with soft tissue malformations

mean

0.0

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

0.00

S.E.

0.00

0.00

0.00

0.00

0.00

percent per litter with skeletal malformations

mean

0.3

0.0

0.0

0.8

0.3

S.D.

1.28

0.00

0.00

2.65

1.46

S.E.

0.26

0.00

0.00

0.53

0.30

total percent per litter with malformations

mean

0.3

0.2

0.4

0.8

0.3

S.D.

1.28

1.20

2.00

2.65

1.46

S.E.

0.26

0.25

0.40

0.53

0.30

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

modified statistics used.

none significantly different from control group

Table S14

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor:EASTMAN CHEMICAL Co.

Summary of fetuses and litters with variations [absolute no.]

Day 20

Dose group:

fetuses

litters

Dose group:

1

2

3

4

5

1

2

3

4

5

number examined externally

322

348

352

348

347

24

24

25

25

24

number with findings

0

0

0

0

0

0

0

0

0

0

number examined viscerally

322

348

352

348

347

24

24

25

25

24

liver- accessory lobule(s)

0

2

4

0

1

0

2

2

0

1

hemorrhagic iris

0

0

1

0

0

0

0

1

0

0

major blood vessel variation

2

2

1

0

0

2

2

1

0

0

haemorrhagic ring around the iris

0

0

0

2

0

0

0

0

1

0

renal papilla(e) not developed and/or distended ureter(s)

2

0

1

0

2

1

0

1

0

2

number examined skeletally

322

348

352

348

347

24

24

25

25

24

sternebra(e)  #5 and/or #6 unossified

23

44

41

39

66

12

13

15

15

16

bent rib(s)

4

3

4

8

7

4

1

3

5

3

14th rudimentary rib(s)

28

22

40

50

64

10

13

17

15

18

reduced ossification of the skull

3

2

3

1

6

2

1

3

1

3

cervical centrum #1 ossified

51

68

53

53

49

19

20

20

17

13

sternebra(e) malaligned(slight or moderate)

3

2

5

1

1

3

2

5

1

1

pubis unossified

0

0

0

1

0

0

0

0

1

0

7th cervical rib(s)

2

4

2

2

9

2

4

2

2

4

sternebra(e)  #1,#2,#3 and/or #4 unossified

0

0

2

0

1

0

0

2

0

1

reduced ossification of the vertebral arches

2

0

1

1

1

2

0

1

1

1

reduced ossification of the 13th rib(s)

0

2

4

0

0

0

2

3

0

0

hyoid unossified

1

0

0

2

0

1

0

0

2

0

2 7 presacral vertebrae

0

0

0

4

1

0

0

0

1

1

bent scapula(e)

0

0

0

1

1

0

0

0

1

1

entire sternum unossified

0

0

0

1

1

0

0

0

1

1

14th full rib(s)

0

0

0

5

0

0

0

0

1

0

vertebral centra unossified

0

0

0

1

0

0

0

0

1

0

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

Table S15

Project No.:WIL-387076

Rat oral prenatal developmental tox study of n-butyl-pyrrolidone

Sponsor:EASTMAN CHEMICAL Co.

Summary of litter proportions of variations % per litter

Day 20

dose group:

1

2

3

4

5

number of litters examined externally

24

24

25

25

24

number of litters with findings

0

0

0

0

0

number of litters examined viscerally

24

24

25

25

24

liver- accessory lobule(s)

mean

0.0

0.7

1.0

0.0

0.3

S.D.

0.00

2.26

3.93

0.00

1.28

S.E.

0.00

0.46

0.79

0.00

0.26

hemorrhagic iris

mean

0.0

0.0

0.3

0.0

0.0

S.D.

0.00

0.00

1.25

0.00

0.00

S.E.

0.00

0.00

0.25

0.00

0.00

major blood vessel variation

mean

0.6

0.5

0.4

0.0

0.0

S.D.

2.03

1.85

2.00

0.00

0.00

S.E.

0.41

0.38

0.40

0.00

0.00

hemorrhagic ring around the iris

mean

0.0

0.0

0.0

0.6

0.0

S.D.

0.00

0.00

0.00

2.86

0.00

S.E.

0.00

0.00

0.00

0.57

0.00

renal papilla(e) not developed and/or distended ureter(s)

mean

0.8

0.0

0.3

0.0

0.6

S.D.

4.08

0.00

1.54

0.00

1.90

S.E.

0.83

0.00

0.31

0.00

0.39

sternebra(e)  #5 and/or #6 unossified

mean

6.7

12.0

11.6

11.3

18.2

S.D.

8.14

15.49

14.34

12.68

21.35

S.E.

1.66

3.16

2.87

2.54

4.36

bent rib(s)

mean

1.4

0.8

1.2

2.6

2.2

S.D.

3.26

4.08

3.76

6.48

6.45

S.E.

0.67

0.83

0.75

1.30

1.32

14th rudimentary rib(s)

mean

9.1

6.3

11.4

14.5

17.4

S.D.

12.38

8.29

12.84

19.57

19.92

S.E.

2.53

1.69

2.57

3.91

4.07

reduced ossification of the skull

mean

1.1

0.6

0.8

0.3

1.7

S.D.

3.93

2.72

2.29

1.67

4.61

S.E.

0.80

0.56

0.46

0.33

0.94

cervical centrum #1 ossified

mean

15.5

19.9

15.1

14.9

13.4

S.D.

13.02

20.84

12.82

15.72

21.49

S.E.

2.66

4.25

2.56

3.14

4.39

sternebra(e) malaligned(slight or moderate)

mean

1.1

0.5

1.6

0.5

0.3

S.D.

3.10

1.82

3.39

2.50

1.28

S.E.

0.63

0.37

0.68

0.50

0.26

pubis unossified

mean

0.0

0.0

0.0

0.3

0.0

S.D.

0.00

0.00

0.00

1.25

0.00

S.E.

0.00

0.00

0.00

0.25

0.00

7th cervical rib(s)

mean

0.6

1.1

0.5

0.6

2.2

S.D.

2.13

2.57

1.70

2.15

6.72

S.E.

0.44

0.53

0.34

0.43

1.37

sternebra(e)  #1,#2,#3 and/or #4 unossified

mean

0.0

0.0

0.5

0.0

0.3

S.D.

0.00

0.00

1.85

0.00

1.46

S.E.

0.00

0.00

0.37

0.00

0.30

reduced ossification of the vertebral arches

mean

0.7

0.0

0.3

0.4

0.3

S.D.

2.25

0.00

1.33

2.22

1.70

S.E.

0.46

0.00

0.27

0.44

0.35

reduced ossification of the 13th rib(s)

mean

0.0

0.6

1.1

0.0

0.0

S.D.

0.00

2.04

3.13

0.00

0.00

S.E.

0.00

0.42

0.63

0.00

0.00

hyoid unossified

mean

0.4

0.0

0.0

0.7

0.0

S.D.

2.04

0.00

0.00

2.54

0.00

S.E.

0.42

0.00

0.00

0.51

0.00

27 presacral vertebrae

mean

0.0

0.0

0.0

1.1

0.2

S.D.

0.00

0.00

0.00

5.33

1.20

S.E.

0.00

0.00

0.00

1.07

0.25

bent scapula(e)

S.E.

0.0

0.0

0.0

0.4

0.3

S.D.

0.00

0.00

0.00

2.22

1.70

S.E.

0.00

0.00

0.00

0.44

0.35

entire sternum unossified

mean

0.0

0.0

0.0

0.4

0.3

S.D.

0.00

0.00

0.00

2.22

1.70

S.E.

0.00

0.00

0.00

0.44

0.35

14th full rib(s)

mean

g.g

g.g

g.g

1.3

g.g

S.D.

g.gg

g.gg

g.gg

g.g7

g.gg

S.E.

g.gg

g.gg

g.gg

1.33

g.gg

vertebral centra unossified

mean

g.g

g.g

g.g

g.4

g.g

S.D.

g.gg

g.gg

g.gg

2.22

g.gg

S.E.

g.gg

g.gg

g.gg

g.44

g.gg

total variations

percent per litter with external variations

mean

0.0

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

0.00

S.E.

0.00

0.00

0.00

0.00

0.00

percent per litter with soft tissue variations

mean

1.4

1.2

2.0

0.6

0.8

S.D.

4.44

2.79

5.52

2.86

2.22

S.E.

0.91

0.57

1.10

0.57

0.45

percent per litter with skeletal variations

mean

32.3

35.5

37.3

41.7

45.1

S.D.

16.86

21.01

21.02

21.49

25.95

S.E.

3.44

4.29

4.20

4.30

5.30

total percent per litter with variations

mean

33.3

36.5

38.6

42.3

45.3

S.D.

16.75

21.09

20.91

21.12

25.57

S.E.

3.42

4.31

4.18

4.22

5.22

1 - 0 mg/kg/day

2 - 200 mg/kg/day

3 - 300 mg/kg/day

4 - 400 mg/kg/day

5 - 500 mg/kg/day

modified statistics used.

none significantly different from control group

Conclusions:
Maternal toxicity was evidenced at 500 mg/kg/day by higher incidences of rales and lower mean body weight gains, body weights, and food consumption during the treatment period. Although test substance-related effects on body weight gains and food consumption were noted at 300 and 400 mg/kg/day and rales were noted at 300 and 400 mg/kg/day, these effects were transient and not considered adverse. Developmental toxicity was noted at 500 mg/kg/day group as evidenced by lower mean fetal body weights. No evidence of developmental toxicity was observed at 200, 300, or 400 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day was considered to be the noobserved-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when n-butyl pyrrolidone was administered orally by gavage to bred Crl:CD(SD) rats.
Executive summary:

The objectives of the study were to determine the potential of the test substance, N-Butylpyrrolidone, to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a noobserved-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity.

The test substance, N-butyl pyrrolidone, in the vehicle (deionized water) was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 200, 300, 400, and 500 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Results

One female in the 500 mg/kg/day group was found dead on gestation day 10. There were no remarkable clinical observations or effects on body weight or food consumption noted for this female prior to death and no remarkable macroscopic findings were noted at necropsy. Therefore, the single death in the 500 mg/kg/day group was not attributed to test substance administration. All other females survived to the scheduled necropsy on gestation day 20. Rales were noted in a dose-related manner in the 300, 400, and 500 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration; however, based on the incidence and frequency of this finding, rales were only considered adverse at 500 mg/kg/day. Clear and red material around the mouth were noted for females in the 500 mg/kg/day group approximately 1 hour following dose administration. These findings were generally noted once per animal and therefore were considered test substance-related, but not adverse. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

A test substance-related lower mean body weight gain and lower mean food consumption were noted for the 500 mg/kg/day group when the overall treatment period (gestation days 6-20) was evaluated due to a mean body weight loss during gestation days 6-9 and lower mean food consumption during the first week of dosing (gestation days 6-12). As a result, mean body weights in the 500 mg/kg/day group were lower (5.0% to 6.0%) than the control group during gestation days 9-15 and a lower mean net body weight gain was observed compared to the control group. Mean net body weight and gravid uterine weight in the 500 mg/kg/day group was comparable to the control group.

Test substance related mean body weight losses and lower mean food consumption were noted in the 200, 300, and 400 mg/kg/day groups immediately following the first dose (gestation day 6-7). Mean body weight gains and food consumption in these groups were comparable to the control group throughout the remainder of the treatment period. Furthermore, mean body weights in the 200, 300, and 400 mg/kg/day groups were unaffected by test substance administration. Therefore, the initial decrements in body weight and food consumption observed in these groups were not considered adverse. Mean net body weight gains in the 300 and 400 mg/kg/day groups were lower than the control group. Mean net body weight gain in the 200 mg/kg/day group and mean net body weights and gravid uterine weights in the 200, 300, and 400 mg/kg/day groups were unaffected by test substance administration.

There were no test substance-related macroscopic findings noted at any dosage level. Although the control group had approximately 4% to 8% fewer mean live fetuses than the test substance-treated groups, these differences were not statistically significant. The mean number of viable fetuses for the concurrent control group (13.4 fetuses) was at the lower end of the WIL Research historical control data, containing data from 80 data sets. Correspondingly, the concurrent control group had the highest mean fetal body weights (4.1 g, 3.9 g, and 4.0 g, for male, female and combined, respectively) compared to the WIL Research historical control data. Test substance-related lower (up to 10%) mean fetal body weights were observed at 500 mg/kg/day. Slight decrements (5%) in mean fetal body weights in the 300 and 400 mg/kg/day groups were considered due to the lower number of fetuses per litter and increased fetal body weights in the concurrent control group. Mean fetal body weights of the 300 and 400 mg/kg/day groups were equal to the mean values in the WIL Research historical control. Therefore, intrauterine growth and survival at 400 mg/kg/day and below were considered unaffected by test substance administration. No test substance fetal malformations or developmental variations were noted at 200, 300, 400, and 500 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study was conducted as part of the product development and commercialization strategy for the United States by a US company. The data is being updated in the REACH dossier to satisfy the data notification requirements, even though this study was not performed for REACH or for entities in the EU.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Identification : n-Butyl Pyrrolidone (also known as TamiSolve® NxG; CAS No. 3470 98-2)
Batch No.: G180445430
Receipt Date: 14 May 2019
Expiration Date: 19 Apr 2020
Physical Description: Clear, colorless liquid
Purity: 99.79%
Water Content: < 0.01%
Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected from light, under nitrogen
Supplier: Chemical Marketing Concepts (US)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Test System
Receipt
On 11 Oct 2019, time-mated female New Zealand White rabbits were received from Envigo Global Services, Inc., Denver, PA on Gestation Day 2, 3, or 4. The animals were approximately 6 months old at receipt and weighed between 2956 and 4426 g on Gestation Day 0.

Justification for Test System and Number of Animals
The New Zealand White rabbit is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the New Zealand White rabbit. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The number of animals selected for this study was based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, August 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, January 2001, which recommend evaluation of approximately 20 females/group with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, 24 females/group was an appropriate number of animals to obtain a sample size of 20 females/group at termination.

Animal Identification
Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system).

Quarantine
After receipt at the Testing Facility, the New Zealand White rabbits were acclimated prior to the initiation of dosing.

Selection, Assignment, and Disposition of Animals
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Animals at extremes of body weight range were not assigned to groups.
The disposition of all animals was documented in the Study Records.

Husbandry
Housing
Animals were individually housed in stainless steel perforated floor cages suspended above appropriate bedding and equipped with an automatic watering valve.
Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number, dosage level, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.
Environmental Conditions
Target temperatures of 61 °F to 71 °F (16 °C to 22 °C) with a relative target humidity of 30 % to 70 % were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms.
Food
PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322 was provided ad libitum throughout the study, except during the acclimation period when food was provided according to Testing Facility SOPs.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Water
Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required.
Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study
Animal Enrichment
Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment or to aid in maintaining the animals’ oral or gastrointestinal health.
Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 7 through 28. All animals were dosed at approximately the same time each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Formulation and Analysis
Preparation of Vehicle

The vehicle, deionized water, was dispensed daily for administration to Group 1 control animals and weekly for preparation of the test substance formulations. The vehicle was stirred continuously during dosing. Details of the dispensing of the vehicle have been retained in the Study Records.

Preparation of Test Substance
Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5 °C), protected from light, under nitrogen, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

Sample Collection and Analysis
Dose formulation samples were collected for analysis.

Analytical Method
Analyses were performed by a high performance liquid chromatography method using ultraviolet absorbance detection using a validated analytical procedure (Engda E. Analytical Validation and Stability Extension Study of n-Butyl Pyrrolidone in Aqueous Formulations (Study No. 00387129). Charles River, Ashland, OH, 2019.)

Concentration Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤ 10 % and if mean sample concentration results were within or equal to ± 15 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability of test substance formulations was established over the range of concentrations used on this study for at least 24 hours at room temperature and for 13 days of room temperature and refrigerated (2 °C to 8 °C) storage in an analytical extension validation study. Therefore, stability of test substance formulations was not assessed on this study.
Details on mating procedure:
N/A, animals were purchased time-mated
Duration of treatment / exposure:
21 days
Frequency of treatment:
Daily from Gestation 7 to 28
Duration of test:
21 days
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were dosed via oral gavage once daily during Gestation Days 7–28.
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy findings, intrauterine growth and survival, and fetal morphology.
Maternal examinations:
Viability
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Observations
The animals were removed from the cage, and a detailed clinical observation was performed once daily, beginning on the day of receipt and lasting through euthanasia. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 1 hour.

Body Weights
Animals were weighed individually on Gestation Days 0 (by supplier), 4, and 7–29.
Gravid uterine weight was collected and corrected body weight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and corrected body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Food Consumption
Food consumption was quantitatively measured on Gestation Days 4–29.

Ovaries and uterine content:
Necropsy
Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

Tissue Collection and Preservation
Gross lesions were collected and preserved in 10 % neutral buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.

Ovarian and Uterine Examinations
The uterus was weighed, and the ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. The placentae were also examined. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.). Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.

External
Each viable fetus was examined in detail, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

Visceral (Internal)
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads from all fetuses were examined by a mid coronal slice.
All carcasses were eviscerated, skinned, and fixed in 100% ethyl alcohol for subsequent examination of skeletons.

Skeletal
Each eviscerated fetus, following fixation in alcohol, was stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.

Statistics:
All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1 % and 5 % levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated by sex and occasion or by litter. Descriptive statistics number, mean, standard deviation, percentage, and/or incidence were reported whenever possible. Calculated values on the Provantis tables may not be reproducible from the individual values presented because all calculations are conducted using non-rounded values. Inferential statistics were performed according to the matrix below when possible, but excluded semi quantitative data, and any group with less than 3 observations. Data obtained from nongravid animals were excluded from statistical analysis.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Dose-dependent, increased incidences of decreased fecal output were observed for 2 and 5 females (1 to 8 occasions each) in the 150 and 300 mg/kg/day groups, respectively, at the daily examinations sporadically during Gestation Days 12–29; a single occurrence of absent fecal output was also noted for 1 of the females in the 300 mg/kg/day group. The excreta-related observations noted in the 300 mg/kg/day group corresponded to the reduced mean food consumption noted in this group. No other test substance related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Abnormal breathing sounds were noted for 1, 5, 3, and 3 females in the control, 50, 150, and 300 mg/kg/day groups, respectively, at the daily examinations and/or postdosing observations sporadically during Gestation Days 10-29. Due to the absence of a dose response and the occurrence in the control group, this observation was attributed to the dosing procedures and not the test substance. Other observations noted in the test substance-treated groups, including fur staining on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
With the exception of Female Nos. 1502, 2523, and 3507 in the control, 50, and 150 mg/kg/day groups, respectively, all females were determined to be gravid.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Description (incidence):
All females in the control, 50, 150, and 300 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 29, including Female No. 2524 in the 50 mg/kg/day group that delivered 1 fetus following euthanasia on Gestation Day 29.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant mean body weight loss was noted in the 300 mg/kg/day group during Gestation Days 7–10 (2.58 % loss) compared to the control group (0.97 % gain). Lower (not statistically significant) mean body weight gains were noted in this group during Gestation Days 10–13 (0.72 % gain compared to 1.54 % gain in the control group) and 13–20 (2.64 % gain compared to 3.78 % gain in the control group), with a statistically significantly higher body weight gain noted during Gestation Days 20–29 (5.69 % gain compared to 2.36 % gain in the control group). The higher mean body weight gain noted at the end of the dosing period was not of sufficient magnitude to negate the early decrements, thus resulting in a lower mean body weight gain (6.45 % gain compared to 8.91 % gain in the control group) when the entire treatment period (Gestation Days 7–29) was evaluated. Although these decrements were not of sufficient magnitude to result in statistically significantly lower mean absolute body weights during the treatment period (0.3 % to 5.6 % lower than the control group), a lower (45.4 %, not statistically significant) corrected body weight gain was noted in this group compared to the control group. Therefore, the body weight effects noted in the 300 mg/kg/day group were considered test substance-related and adverse.

Mean corrected body weight and gravid uterine weight in the 300 mg/kg/day group were generally comparable to the control group; no statistically significant differences were noted.

Lower (not statistically significant) mean body weight gains were noted in the 50 and 150 mg/kg/day groups following the initiation of dosing (Gestation Days 7–10), which were considered test substance-related but not adverse. Mean absolute body weights, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 150 mg/kg/day groups were generally comparable to the control group; no statistically significant differences were noted.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption, evaluated as g/animal/day, in the 300 mg/kg/day group was statistically significantly lower than the control group during Gestation Days 7–10 (48.82 %), 10 13 (47.45 %), and 13–20 (34.79 %), as well as when the entire treatment period (Gestation Days 7–29; 26.44 %) was evaluated. These decrements corresponded to the aforementioned body weight effects.
Mean maternal food consumption in the 50 and 150 mg/kg/day groups were unaffected by test substance administration and was generally comparable to the control group throughout the study. Any statistically significant differences from the control group were transient, had no corresponding effect on absolute body weight/body weight gain, and did not occur in a dose-related manner.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on Gestation Day 29, no test substance-related internal findings were observed at dosage levels of 50, 150, and 300 mg/kg/day. Macroscopic findings observed in the test substance treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.
Changes in postnatal survival:
not examined
Description (incidence and severity):
Not part of this study design as there was not parturition.
External malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related external malformations observed in fetuses in this study. External malformations were observed in 1 fetus each in the control and 150 mg/kg/day groups. Fetus No. 3503-05 in the 150 mg/kg/day group was noted with an inwardly rotated right hindlimb. This finding was not considered test substance-related because it occurred in a single fetus in the mid dosage group. Fetus No. 1508-09 in the control group was noted with cleft lower mandible (medial), cleft upper lip (medial), short snout, cleft palate (entire length), and split tongue.
No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related skeletal malformations observed in fetuses in this study. Skeletal malformations were observed in 1(1), 0(0), 3(2), and 1(1) fetuses (litters) in the control, 50, 150, and 300 mg/kg/day groups, respectively.
Fetus No. 4503-01 in the 300 mg/kg/day group had 8 lumbar vertebrae, fused lumbar centra, and a lumbar hemivertebra. In the 150 mg/kg/day group, Fetus No. 3506-03 was noted with 8 lumbar vertebrae, Fetus No. 3523-04 was noted with 6 lumbar vertebrae, and Fetus No. 3523 06 was noted with branched ribs, 8 cervical vertebrae, 11 thoracic vertebrae, and a thoracic hemivertebra. These findings were not considered test substance-related due to the absence of a dose response and the mean litter proportions of the individual findings were not statistically significantly different from the control group. In the control group, Fetus No. 1508 09 was noted with misshapen right mandible and premaxilla bones and split palatine bones, which correlated to the cleft malformations or short snout observed externally; fused ventral arch atlas, cervical centra, and thoracic centra were also noted for this fetus.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, and/or were not observed in a dose-related manner.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related visceral malformations observed in fetuses in this study. Visceral malformations were observed in 3(3), 0(0), 1(1), and 2(2) fetuses (litters) in the control, 50, 150, and 300 mg/kg/day groups, respectively.
In the 300 mg/kg/day group, Fetus No. 4506-01 had an absent right thyroid gland and Fetus No. 4508-03 was noted with a retroesophageal right subclavian artery. Fetus No. 3518-08 in the 150 mg/kg/day group was also noted with a retroesophageal right subclavian artery. These findings were not considered test substance-related due to the absence of a dose response and the finding was noted similarly in the control group (Fetus No. 1519-04). Fetus No. 1508 09 in the control group was noted with a diaphragmatic hernia; this fetus also had multiple external malformations. Also, in the control group, Fetus No. 1521 05 was noted with a caudally malpositioned right kidney.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, and/or were not observed in a dose-related manner.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 210(23), 194(23), 211(23), and 216(24) in the control, 50, 150, and 300 mg/kg/day groups, respectively. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other:
Remarks:
No adverse effects found
Abnormalities:
no effects observed
Developmental effects observed:
no

Summary data tables are attached in the "background material" section as a single .PDF.

Conclusions:
Based on adverse maternal effects, including body weight loss, reduced food consumption, and corresponding excreta-related clinical observations at 300 mg/kg/day, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when N-Butylpyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits. Intrauterine growth and survival and fetal morphology were unaffected by test substance exposure at all dosage levels. Therefore, 300 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity.
Executive summary:

The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

All females survived to the scheduled necropsy on Gestion Day 29, including 1 female in the 50 mg/kg/day group that delivered on Gestation Day 29. Test substance-related, dose-dependent incidences of decreased fecal output were observed in the 150 and 300 mg/kg/day groups at the daily examinations; this observation corresponded to the reduced mean food consumption noted in the 300 mg/kg/day group. No other test substance-related clinical observations were noted at any dosage level at the daily examinations or approximately 1-hour following dose administration.

Test substance-related mean body weight losses following the initiation of treatment (Gestation Days 7–10) and lower mean body weight gains during Gestation Days 10-13 and 13-20 were noted in the 300 mg/kg/day group, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated. Mean food consumption in this group was also lower than the control group throughout the treatment period. Although the decrements in this group were not of sufficient magnitude to result in noteworthy changes in mean absolute body weights throughout the treatment period, a lower corrected body weight gain was noted in this group compared to the control group; therefore, the body weight effects noted in the 300 mg/kg/day group were considered adverse. Mean corrected body weights and gravid uterine weights in this group were comparable to the control group. In the 50 and 150 mg/kg/day groups, test substance-related lower mean body weight gains, compared to the control group, were also noted following the initiation of treatment (Gestation Days 7–10). Mean body weight gains in these groups were comparable to the control group for the remainder of the treatment period, as well as when the overall treatment period was evaluated. In addition, mean absolute body weights, food consumption, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 150 mg/kg/day groups were comparable to the control group. Therefore, the initial decrements in these groups were considered nonadverse.

No test substance-related internal findings were observed at any dosage level at the scheduled necropsy on Gestation Day 29.

Intrauterine growth and survival were unaffected by test substance administration at all dosage levels. There were also no test substance-related effects on fetal morphology in the 50, 150, and 300 mg/kg/day groups.

In conclusion, based on adverse maternal effects, including body weight loss, reduced food consumption, and corresponding excreta-related clinical observations at 300 mg/kg/day, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when N-Butylpyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits. Intrauterine growth and survival and fetal morphology were unaffected by test substance exposure at all dosage levels. Therefore, 300 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Species:
rat
Quality of whole database:
The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
600 mg/m³
Species:
rat
Quality of whole database:
The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Species:
rat
Quality of whole database:
The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.
Additional information

There are four reliable developmental toxicity studies available for inhalation ( 1 in rats), oral (1 in rats, 1 in rabbits)and dermal routes of exposure (1 in rats).

In an inhalation developmental toxicity study (Charles River Laboratories, 2016c; Report No. WIL – 387081), the test substance was administered via whole-body inhalation exposure to 3 groups of 24 bred female Crl:CD(SD) rats for 6 hours per day from Gestation Days 6–19. Based on a range-finding study in nonpregnant female rats, target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). Maternal toxicity was evidenced by adverse clinical observations and mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption in the 1.2 mg/L group throughout the gestation treatment period. Developmental effects were noted at 1.2 mg/L as evidenced by lower mean fetal weights, which correlated with a lower mean gravid uterine weight and decrements in maternal food consumption and body weight gain during the fetal period (Gestation Days 15-20). Lower mean fetal weights were also noted in the 0.3 and 0.6 mg/L groups; however, mean fetal weight values in these groups were greater than the minimum mean values and only slightly lower than the mean and median values in the test facility historical control data. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the test facility historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups, and were not considered test substance-related. Test substance-related fetal skeletal developmental variations (higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified) were noted in the 1.2 mg/L group, which corresponded with the lower mean fetal weights noted in this group. No adverse maternal or developmental effects were noted at 0.3 and 0.6 mg/L. Based on these results, an exposure level of 0.6 mg/L was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance was administered via whole-body inhalation exposure to bred Crl:CD(SD) rats. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively.

In an oral developmental toxicity study (OECD 414; Charles River Laboratories, 2016a; Report No. WIL - 387076), the test substance was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 200, 300, 400, and 500 mg/kg/day. Maternal toxicity was evidenced at 500 mg/kg/day by higher incidences of rales and lower mean body weight gains, body weights, and food consumption during the treatment period. Although test substance-related effects on body weight gains and food consumption were noted at 300 and 400 mg/kg/day and rales were noted at 300 and 400 mg/kg/day, these effects were transient and not considered adverse. Developmental toxicity was noted at 500 mg/kg/day group as evidenced by lower mean fetal body weights. No evidence of developmental toxicity was observed at 200, 300, or 400 mg/kg/day. Test substance-related lower (up to 10%) mean fetal body weights were observed at 500 mg/kg/day. Slight decrements (5%) in mean fetal body weights in the 300 and 400 mg/kg/day groups were considered due to the lower number of fetuses per litter and increased fetal body weights in the concurrent control group. Mean fetal body weights of the 300 and 400 mg/kg/day groups were equal to the mean values in the WIL Research historical control. Therefore, intrauterine growth and survival at 400 mg/kg/day and below were considered unaffected by test substance administration. No test substance fetal malformations or developmental variations were noted at 200, 300, 400, and 500 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when N-butyl pyrrolidone was administered orally by gavage to bred Crl:CD(SD) rats.

In an oral developmental toxicity study (OECD 414, CHarles River Laboratories; 2020; Report No. WIL-), the test substance was administered orally by gavage to rabbits to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. All females survived to the scheduled necropsy on Gestion Day 29. Test substance-related, dose-dependent incidences of decreased fecal output were observed in the 150 and 300 mg/kg/day groups at the daily examinations; this observation corresponded to the reduced mean food consumption noted in the 300 mg/kg/day group. No other test substance-related clinical observations were noted at any dosage level at the daily examinations or approximately 1-hour following dose administration.

Test substance-related mean body weight losses following the initiation of treatment (Gestation Days 7–10) and lower mean body weight gains during Gestation Days 10-13 and 13-20 were noted in the 300 mg/kg/day group, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated. Mean food consumption in this group was also lower than the control group throughout the treatment period. Although the decrements in this group were not of sufficient magnitude to result in noteworthy changes in mean absolute body weights throughout the treatment period, a lower corrected body weight gain was noted in this group compared to the control group; therefore, the body weight effects noted in the 300 mg/kg/day group were considered adverse. Mean corrected body weights and gravid uterine weights in this group were comparable to the control group. In the 50 and 150 mg/kg/day groups, test substance-related lower mean body weight gains, compared to the control group, were also noted following the initiation of treatment (Gestation Days 7–10). Mean body weight gains in these groups were comparable to the control group for the remainder of the treatment period, as well as when the overall treatment period was evaluated. In addition, mean absolute body weights, food consumption, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 150 mg/kg/day groups were comparable to the control group. Therefore, the initial decrements in these groups were considered nonadverse.

No test substance-related internal findings were observed at any dosage level at the scheduled necropsy on Gestation Day 29.

Intrauterine growth and survival were unaffected by test substance administration at all dosage levels. There were also no test substance-related effects on fetal morphology in the 50, 150, and 300 mg/kg/day groups.

In conclusion, based on adverse maternal effects, including body weight loss, reduced food consumption, and corresponding excreta-related clinical observations at 300 mg/kg/day, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when N-Butylpyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits. Intrauterine growth and survival and fetal morphology were unaffected by test substance exposure at all dosage levels. Therefore, 300 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity.

In a dermal developmental toxicity study (OECD 414; Charles River Laboratories, 2016b; Report No. WIL - 387079), the test substance was administered by dermal application to 3 groups of bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Animals were exposed to the test substance for approximately 23 hours each day. Exposure levels were 375, 500, and 750 mg/kg/day. All females in the control, 375, 500, and 750 mg/kg/day groups survived to the scheduled necropsy. A slightly increased incidence of red material around the eyes was noted in all test substance-treated groups generally throughout the treatment period at the daily examinations; however, given the low occurrence of this finding (generally 1-4 occurrences per animal) and the inability of the animals to groom while outfitted with the Elizabethan collars, this finding was considered nonadverse. No other test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration in the 375, 500, and 750 mg/kg/day groups. No dermal findings were noted at any dosage level. Adverse mean body weight losses and lower mean body weight gains with corresponding reduced mean food consumption were noted in the 750 mg/kg/day group, with the greatest magnitude of change occurring during the fetal period, which resulted in lower mean body weights, net body weight, net body weight gain, and gravid uterine weight in this group. The decrements in body weight and food consumption parameters at 750 mg/kg/day noted during the period of greatest fetal growth (gestation days 15-20) resulted in lower mean fetal weights in this group. Intrauterine growth at 375 and 500 mg/kg/day and survival at 375, 500, and 750 mg/kg/day were unaffected by test substance administration. There were no test substance-related external, visceral, and skeletal malformations or developmental variations noted at any dosage level. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when N-butyl pyrrolidone was administered by dermal application to bred Crl:CD(SD) rats.

In a supporting oral developmental toxicity study the doses were determined in a range-finding study (LPT, 2013d; Report No. 29177). Dose levels of 5, 50 or 500 mg/kg bw/day had been administered to female rats, by gavage from the 6th to 19th day of pregnancy. No test item-related influences were noted at the low and intermediate dose level (5 and 50 mg/kg bw/day) concerning the behavioural toxicity, body weight, food and water consumption, uterus and carcass weights, the examination at autopsy and the parameters of reproduction (number of corpora lutea, implantation sites, resorptions, percentage of pre- and post implantation loss). At the high dose level (500 mg/kg b.w./day) piloerection was noted in 2 of 3 dams and laboured breathing in one dam. Starting of treatment resulted in a distinct reduction in food consumption and a decline in body weight. A total loss of implantation sites was noted in 1 of the 3 high dose dams, whereas the other 2 showed no influence on the parameters of reproduction. The foetuses from the dams of the low and intermediate dose group (5 and 50 mg/kg bw/day) showed no reduction in body weight and no malformations or variations were found during the external examination. In the high dose group (500 mg /kg bw/day), the foetuses from the 2 dams showed a slightly reduced body weight. Again, no malformations or variations were found during the external examination. Therefore, the dose levels for the main study were 5, 50 and 500 mg/kg bw.

In the supporting main study (OECD 414; LPT, 2013c; Report No. 29197), the test substance was administered to female CD® / Crl:CD (SD) rats also at dose levels of 5, 50 or 500 mg/kg bw/day (administration volume: 5 mL/kg b.w./day), orally by gavage from the 6th to 19th day of pregnancy. For dams, the NOAEL was determined as 50 mg/kg b.w./day. At the high dose group (500 mg/kg bw/day), the following observations were made: a slight reduction in body weight and a more distinct reduction in the net and absolute body weight change were noted during the treatment period. After the start of treatment a reduction in food consumption was noted in the high dose group for 2 days. Thereafter, normal level of food intake was reached. In addition, slight reductions of the gravid uterus weight and the carcass weight were noted. No test substance-related influence was noted on the reproductive parameters of the dams. For foetal organisms, the NOAEL was set to > 500 mg/kg b.w./day. The external / internal, skeletal and soft tissue examinations revealed no malformations. No test substance-related increase was noted in the incidence of external / internal, skeletal and soft tissue variations or the skeletal retardations. No dead foetuses and no test substance-related differences in the placental and foetal weights, the number of corpora lutea, implantation sites, resorptions and in the number of runts were noted in the treatment groups. The percentages of pre- and post-implantation loss were also not influenced by the test item.

Even though the study itself is considered reliable and valid, the results of the study are considered not to allow a meaningful interpretation of the results due to the selection of dose levels based upon differences of a factor of ten (log order). The dose range finder had already shown only minimal changes at 500 mg/kg/day in both the fetus and the dams, which suggested that the NOAEL would be around 500 mg/kg/day. Therefore, a different dose level spacing would have been indicated. A further reason to consider the results to be inappropriate for interpretation, is the absence of external, internal or visceral malformations in any of the rats, while the strain of Sprague Dawley rats has a background malformation rate of around 0.5 - 0.8 %. Finally, although there was a decrease in placental weights in male, female and combined conceptuses and a decrease in female fetal body weight; the authors interpreted these changes as not related to treatment (non-adverse) based upon comparison to historical control values. The authors argument is that although the 11.3% reduction in placental weight (p ≤ 0.01) and 8.8% reduction in female fetal weight (p ≤ 0.01) in the 500 mg/kg bw/day group were present, the values were within the historical control (HC) range for the laboratory. Problems with this argument include 1) the concurrent control group is the appropriate value for analysis and unless a problem can be demonstrated with the concurrent control group value, the comparison to HC values should be of minor consideration and 2) where within the HC range of values the treated group falls should be considered. In this instance, the concurrent control values pretty much fall almost exactly mid-range of the HC values, indicating that the concurrent control is producing values that are normal for this strain or rat in this laboratory. In addition, the value for the female fetal body weight (3.1 grams) is at the extreme low end of the HC values. The same can be considered for the male fetal body weights and the combined fetal body weights for the 500 mg/kg bw/day group, although these values were not statistically significantly different from the concurrent control. All of these factors appear to discredit the reasoning for considering these findings as “not related to treatment”. Therefore, a NOAEL of 50 mg/kg bw has to be established for general toxicity because of reduced body weight and body weight gains of the dams and reduced food consumption and gravid uterine weights in the highest dose group.

In conclusion, even though, the data itself is valid, the poor choice of doses made a meaningful interpretation impossible.

The results of the dose range-finding study (OECD 414; LPT, 2013d; Report No. 29177) are also considered not to allow a meaningful interpretation due to the same reasons. The low value of 50 mg/kg bw are overestimating greatly the actual NOAEL, which would have been identified using a more appropriate dose spacing regime.

Justification for classification or non-classification

The fertility and developmental parameters were not affected by test substance administration at all dose levels tested in the OECD 414 developmental toxicity studies in rats and rabbits. NOAEL for maternal toxicity was established at the same dose levels of general systemic toxicity and developmental toxicity. Therefore, reproductive toxicity observed at the highest dose levels is considered to represent a secondary non-specific consequence of other toxic effects of the test substance administration. Additionally, in the 90-day study, no adverse effects were detected during the oestrous cycle assessment and semen parameters assessment. There were no abnormalities detected in reproductive organs, their weights and histopathology. Based on the results of the 90-day study, the highest dose level of 500 mg/kg bw may be regarded as NOAEL for reproductive function. Based on these data, N-Butylpyrrolidone does not need to be classified and labelled as reproductive or developmental toxicant according to Regulation (EC) 1272/2008.

Additional information