1-butylpyrrolidin-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-01-16 to 2014-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the REACH.

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

Test material

Test material information

Constituent 1

Reference substance name:

1-butylpyrrolidin-2-one

EC Number:

222-437-8

EC Name:

1-butylpyrrolidin-2-one

Cas Number:

3470-98-2

Molecular formula:

C8H15NO

IUPAC Name:

1-butylpyrrolidin-2-one

Test material form:

other: liquid

Details on test material:

- Substance type: organic
- Physical state: Clear colorless liquid
- Purity test date: 2013-10-29
- Expiration date of the lot/batch: 01 December 2016
- Stability under test conditions: The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. 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.
- Storage condition of test material: room temperature in the dark
- date received: 01 November 2013

Test animals

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).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral 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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analyzed 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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, 2013; 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, labored 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.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral 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 color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, 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.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination of treatment (during Week 12).
- Dose groups that were examined: all control and high dose animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 90). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: on all animals from each test and control group.
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (Day 90). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Animals fasted: No
- How many animals: on all animals from each test and control group.
- Parameters checked in table [No.3] were examined.

URINALYSIS: Yes (in the context of detailed clinical observations)
- Time schedule for collection of urine: prior to the start of treatment and at weekly intervals thereafter
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organs were weighed: adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, uterus, liver.
HISTOPATHOLOGY: Yes (see table 4)
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.

Other examinations:

- Oestrous Cycle:
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 estrus was recorded.
- Semen assessment:
(i)The left testis and epididymis were removed, dissected from connective tissue and weighed separately. (ii) For the testis, numbers of homogenisation resistant spermatids were analysed in the homogenates from testicular tissue. (iii) For the epididymis, the distal region was incised and a sample of the luminal fluid collected 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. (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 homogentate 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.

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 analyzed 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 analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANOVA with appropriate covarities. Any transformed data were analyzed 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 analyzed 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)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

statistically significant changes without toxicological importance

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

statistically significant changes without toxicological importance

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

intergroup differences were considered to be of no toxicological importance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased liver weights in animals of either sex treated with 500 mg/kg bw and in males treated with 100 mg/kg bw. Males treated with 500 and 100 mg/kg bw/day also showed a statistically significant increase in absolute and relative kidney weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

eight males had an enlarged liver at 500 mg/kg bw, the other findings are without toxicological importance.

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).

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
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 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.

WATER CONSUMPTION
Daily visual inspection of water bottles did not reveal any overt differences in water consumption at 10, 100 or 500 mg/kg bw/day

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ocular effects.

HAEMATOLOGY
There were no toxicologically significant effects of treatment on the haematology parameters measured.
Males treated with 500 mg/kg bw/day had a statistically significant increase in neutrophil and platelet counts and a statistically significant reduction in clotting time. Females treated with 500 mg/kg bw/day had a statistically significant reduction in haemoglobin and haematocrit and a statistically significant increase in neutrophil count. A true dose response was not evident and in the absence of any histopathological correlates, the intergroup differences were considered not to be toxicologically significant.

CLINICAL CHEMISTRY
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 and alkaline phosphatase and a statistically significant increase in cholesterol, bilirubin and bile acid.
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.
Females treated with 500 mg/kg bw/day showed a statistically significant reduction in chloride concentration whilst males treated with 100 mg/kg bw/day showed a statistically significant increase in chloride concentration. The majority of individual values were within normal background ranges and as such were considered not to be of toxicological importance.

NEUROBEHAVIOUR
There were no toxicologically significant changes in the behavioural parameters measured. One male treated with 500 mg/kg bw/day had noisy respiration during the behavioural assessments on Days 48 and 55. Observations of this nature are often experienced as a result of aspiration of a slightly irritant test item formulation and in isolation, these observations were considered not to represent a systemic effect of toxicity. Inter and intra group differences in urination and defecation were considered to represent normal biological variation for rats of the strain and age used.

There were no toxicologically significant changes in functional performance. Animals of either sex treated with 500 mg/kg bw/day had statistically significantly lower overall activity. Females treated with 500 mg/kg bw/day also had a statistically significant reduction in the last 20% of activity. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological importance.

There were no toxicologically significant changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

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.

HISTORICAL CONTROL DATA (attached to the study report). Incidental findings were within historical control data.

OTHER FINDINGS
- Oestrous Cycle assessment: No adverse effects were detected during the oestrous cycle assessments. All control and treated females showed evidence of oestrus.

- Sperm analysis: There were no toxicologically significant effects detected in sperm concentration, morphological assessments or in homogenisation-resistant spermatid counts (see section 7.8.1 for details).

Effect levels

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.

2

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 no adverse effects were observed at 100 and 500 mg/kg bw.

3

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: treatment related, but no adverse effects were observed at 500 mg/kg bw.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Discussion

Administration of the test substance by oral gavage at dose levels of 10, 100 and 500 mg/kg bw/day for 90 consecutive days resulted in treatment related effects at 500 mg/kg bw/day in animals of either sex and in males at 100 mg/kg bw/day.

There were no adverse effects detected in clinical signs, body weight development or food consumption in treated animals. Eight males treated with 500 mg/kg bw/day were found to have an enlarged liver at necropsy. 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. Microscopic examination of the livers of these animals revealed hypertrophy or centrilobular hypertrophy in seven males and all females treated with 500 mg/kg bw/day and three males treated with 100 mg/kg bw/day. These findings are suggestive of a response to mixed function oxidase induction and in the absence of other pathological changes are considered to be adaptive and of limited toxicological significance. These adaptive changes were considered to be responsible for the blood chemical changes apparent; males treated with 500 mg/kg bw/day showed a statistically significant increase in total protein 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 and alkaline phosphatase and a statistically significant increase in cholesterol, bilirubin and bile acid.

Males treated with 500 and 100 mg/kg bw/day had a statistically significant increase in kidney weights, both absolute and relative to terminal body weight. Males treated with 500 mg/kg bw/day had a statistically significant increase in creatinine and calcium. Microscopic assessment of the kidneys revealed 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 treated with 100 and 500 mg/kg bw/day. This finding correlated with the increase in organ weight noted in the males and is considered to be specific to the male rat and does not represent a risk to humans, therefore, the associated changes in calcium and creatinine were also considered not to be of toxicological significance.

Thymic 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. The thymic atrophy noted in both sexes at the high dose is likely to be a secondary stress related response and does not represent an adverse effect of treatment.

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. The increased vacuolation in the adrenal gland cortex may be directly related to the administration of the test substance, however it occurred only in males and it was considered to be related to changes in lipid metabolism and may be linked to the adaptive liver changes observed.

Applicant's summary and conclusion

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 all dose levels tested. 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.