2-Oxepanone, polymer with 1,4-butanediol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 September 2016 - 02 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

dose range finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 July 2016 - 16 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: The toxicity of CAPA 2043, after daily oral administration (gavage) to rats, was investigated over a period of 2 consecutive weeks.
- Short description of test conditions: Three groups, each of 4 male and 4 female Sprague Dawley SD rats, received the test item at dose levels of 100, 300 and 1000 mg/kg/day for 2 consecutive weeks. A fourth similarly constituted group received the vehicle alone (corn oil) and acted as a control.
- Parameters analysed / observed: clinical signs, body weight and food consumption, haematology and clinical chemistry, post mortem organ weights and macroscopic observations.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identity: CAPA 2043
Label name: CAPA(TM) 2043
Chemical name: 2-oxepanone, polymer with 1,4-butanediol
Batch number: WAC000478
CAS number: 31831-53-5
EC number: 608-670-1
Expiry date: 10 May 2018
Appearance: Clear, colourless liquid
Storage conditions: Cool, dry conditions in well-scaled receptacles

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS s.r.l., San Pietro al Natisone (UD), Italy
- Age at arrival: 27-29 days old
- Weight at arrival: 75-99 g
- Housing: up to 5 of one sex in a cage
- Diet (e.g. ad libitum): Laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy); ad libitum
- Water (e.g. ad libitum): Drinking water; ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally, by gavage at a dose volume of 5mL/kg. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The required amount of CAPA 2043 was suspended in the vehicle. The formulations were prepared daily (concentrations of 20, 60 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied. The formulations were prepared daily (concentrations of 20, 60 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

- VEHICLE: corn oil

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

n.a.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

Once a day, 7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle (corn oil) control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

Three groups, each of 4 male and 4 female Sprague Dawley SD rats, received the test item at dose levels of 100, 300 and 1000 mg/kg/day for 2 consecutive weeks. A fourth similarly constituted group received the vehicle alone (corn oil) and acted as a control.
-Dose selection rationale: not reported
- Rationale for animal assignment: Each group comprised 4 male and 4 female rats. The group identification and animal numbers assigned to the treatment are summarized below:
Group 1 (0 mg/kg bw/day) Rat numbers: males (even) 2-8, females (odd) 1-7
Group 2 (100 mg/kg bw/day) Rat numbers: males (even) 10-16, females (odd) 9-15
Group 3 (300 mg/kg bw/day) Rat numbers: males (even) 18-24, females (odd) 17-23
Group 4 (1000 mg/kg bw/day) Rat numbers: males (even) 26-32, females (odd) 25-31
The rat numbers listed above formed the last digits of a computer generated 8 figure animal number (the remaining digits of the animal number were different for each concurrent study and served to ensure unique animal numbering for any study employing computerised data collection). The computerised system used in this study was Pristima, Version 6.4.1.

Positive control:

n.a.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday, a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during treatment, each animal was observed, and any clinical signs recorded. Observations were performed at the same time interval each day; the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, twice weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of week 2
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: all surviving male and female rats from each group
- Parameters checked in table No.1 in box “Any other information on materials & methods” were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of week 2
- Animals fasted: Yes
- How many animals: all surviving male and female rats from each group
- Parameters checked in table No.2 in box “Any other information on materials & methods” were examined.

OTHER: Organ weights: spleen, testes, thymus (where present)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see table 3 in box "Any other information on materials & methods incl. tables"

HISTOPATHOLOGY: No

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs in the surviving animals were limited to decreased activity and prone posture, observed on Day 6 in a single male animal of the high dose group (1000 mg/kg/day).
Lethargy, piloerection and prone posture were seen in one female of the same group on Day 11.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two male animals of the high dose group, were found dead on Day 11 of the study. One animal was cannibalised by cage mates, while no signs were oberved during the in vivo phase in athe other animal. Post mortem macroscopic examination showed enlarged liver and distended urinary bladder in the latter animal. These changes were not considered to clearly establish the cause of death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Very slight but statistically significant reductions in body weight were seen in the high dose males on Days 8 and 11 (-6% and -7%, respectively). No significant differences were observed in the females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly reduced in the high dose males on Days 8 and 15 (-14% and -22%), respectively when compared to controls. No significant differences in food consumption were observed in the females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to controls, erythrocytes were slightly higher in males dosed at 1000 mg/kg/day (9%). Due to the minimal severity and direction, this change was considered of no toxicological significance.
In addition, platelets were increased in males dosed at 100 and 300 mg/kg/day (40% and 48%, respectively) and decreased in one female receiving 300 mg/kg/day (95%). Due to the absence of dose-relation, those findings were considered incidental.
Coagulation: No changes were recorded.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to controls, glucose was increased in animals dosed at 1000 mg/kg/day (86% in males, 50% in females). The severity of this finding and the absence of other related changes were not considered to be suggestive of tissue/organ injury.
Other fluctuations were recorded, such as: decrease of urea in males dosed at 100 and 300 mg/kg/day (22% and 23%, respectively), increase of calcium in those receiving 300 and 1000 mg/kg/day (4%, both groups) and increase of alanine aminotransferase and aspartate aminotransferase in one male (300 mg/kg/day, 3.4 and 2.6-fold, respectively). Due to the absence of dose-relation and/or the minimal severity, these findings were considered of no toxicological relevance.

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 changes of toxicological significance were seen in the weight of the organs.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animals killed at termination did not show any relevant macroscopic changes.
The changes observed such as multiple red areas in the thymus are suggested to be incidental in untreated Sprague Dawley SD rats of the same age.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

Two male animals dosed at 1000 mg/kg/day were found dead on Day 11 of the study. One of the 2 animals was cannibalised by cage mates. No signs were observed during the in vivo phase in the second one. Changes observed at post mortem observations were not considered to clearly establish the cause of death. Clinical signs (lethargy/decreased activity, prone posture and/or piloerection) were occasionally observed in individual animals dosed at 1000 mg/kg/day. Slight reductions in body weight, terminal body weight and food consumption were also observed during the in vivo phase in the males dosed at 1000 mg/kg/day. Increases in glucose and other minor fluctuations of clinical chemistry parameters were seen in animals dosed at 1000 mg/kg/day. No changes of toxicological significance were observed at post mortem observations (organ weights and macroscopic observations).

For summary of relevant finding see table 4 in box "Any other information on results incl. tables".

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Critical effects observed:

not specified

Table 4: Summary of relevant findings in the 2 -week study with CAPA 2043

	Males				Females
mg/kg bw/day	0	100	300	1000	0	100	300	1000
n	4	4	4	4	4	4	4	4
Mortality	-	-	-	2	-	-	-	-
Clinical signs	-	-	-	1 animal: Cannibalized, Muzzle; Cannibalized, Ventrum 1 animal: Decreased activity, slight prone	-	-	-	1 animal: Lethargy, Piloerection, Moderate Prone 1 animal: Abrasion, Tail, Slight
Mean body weight (g) Dosing Day 1/4/8/11/15	185.98/209.28/236.00/254.10/259.63	186.43/208.70/237.60/261.50/266.95	187.00/210.00/240.80/264.35/268.73	143.03/186.08/201.90/221.03*D/235.70/241.75	150.88/159.83/172.88/185.18/180.38	147.20/157.65/170.65/184.28/181.10	147.48/156.48/168.73/180.73/179.98	146.33/154.13/165.33/175.75/173.88
Mean food consumption (g) Dosing Day 6/8/15	19.46/20.09/17.63	19.25/20.66/18.44	19.33/20.87/18.99	19.25/17.29/13.78	15.81/13.72/12.48	14.32/13.79/12.33	14.26/14.13/12.43	13.81/13.11/11.84
Haematology
Erythrocytes (x10^6/µL)	6.890	6.738	6.913	7.515*D	7.103	6.878	7.270	7.203
Platelets (x10^3/µL)	777.7	1089.0*	1151.3*D	940.0	1114.3	1124.5	783.0	1109.3
Clinical Chemistry
Glucose (mg/dL)	60.23	49.55	63.83	111.90+D	62.90	65.50	87.78	94.23*D
Ca (mMmol/L)	2.535	2.598	2.638+D	2.635*D	2.578	2.565	2.553	2.650
Urea (mg/dL)	33.63	26.28+D	25.83+D	30.30	36.60	36.38	38.43	32.15
Alanine aminotransferase (U/L)	41.18	43.73	66.58	64.15	33.58	30.60	34.20	33.70
Aspartate aminotransferase (U/L)	91.83	84.80	120.85	73.35	84.08	67.48	69.38	67.90
Gross pathological findings
	1 animal: Thymus red - left lobe	1 animal: liver: single, pale	1 animal: Thymus multiple red - right lobe	1. animal (unscheduled death; cannibalised animal): Thymus: Multiple, Dark red 2. animal /unscheduled death): Epididymis small, liver enlarged, prostate gland small, seminal vesicles small, urinary bladder distended) 1 animal (final sacrifice): Thymus multiple red - right lobe	-	1 animal: Uterus distended, clear, fluid	-	-

* Dunnett LSD Test Significant at the 0.05 level

+D Dunnett LSD Test Significant at the 0.01 level

Conclusions:

In conclusion, the test item CAPA 2043, when orally administered at dose levels of 100, 300 and 1000 mg/kg/day, resulted to be satisfactorily tolerated by Sprague Dawley rats at the low and intermediate dose levels (100 and 300 mg/kg/day), while the highest dose level of 1000 mg/kg/day induced some toxicity (mortality or occasional clinical signs, slight reductions in body weight and food consumption). Therefore, it can be concluded that 300 mg/kg/day was the NOEL (No Observed Effect Level) in this study. The highest dose of 1000 mg/kg/day was not considered to be safe in a subsequent toxicity study of longer duration in Sprague Dawley rats.

Executive summary:

In a 2-week repeated dose toxicity study, the test item CAPA 2043 (2-oxepanone, polymer with 1,4-butanediol) in corn oil was administered to Sprague-Dawley rats (4/sex/dose) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day for once a day, 7 days a week for 2 weeks.

Two male animals dosed at 1000 mg/kg/day were found dead on Day 11 of the study. One of the 2 animals was cannibalised by cage mates. No signs were observed during thein vivophase in the second one. Changes observed at post mortem observations were not considered to clearly establish the cause of death. Clinical signs (lethargy/decreased activity, prone posture and/or piloerection) were occasionally observed in individual animals dosed at 1000 mg/kg/day. Slight reductions in body weight, terminal body weight and food consumption were also observed during thein vivophase in the males dosed at 1000 mg/kg/day. Increases in glucose and other minor fluctuations of clinical chemistry parameters were seen in animals dosed at 1000 mg/kg/day. No changes of toxicological significance were observed atpost mortemobservations (organ

weights and macroscopic observations). In conclusion, the test item CAPA 2043, when orally administered at dose levels of 100, 300 and 1000 mg/kg/day, resulted to be satisfactorily tolerated by Sprague Dawley rats at the low and intermediate dose levels (100 and 300 mg/kg/day), while the highest dose level of 1000 mg/kg/day induced some toxicity (mortality or occasional clinical signs, slight reductions in body weight and food consumption). Therefore, it can be concluded that 300 mg/kg/day was the NOEL (No Observed Effect Level) in this study. The highest dose of 1000 mg/kg/day was not considered to be safe in a subsequent toxicity study of longer duration in Sprague Dawley rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

21 September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Oxepanone, polymer with 1,4-butanediol

Cas Number:

31831-53-5

IUPAC Name:

2-Oxepanone, polymer with 1,4-butanediol

Constituent 2

Reference substance name:

CAPA 2043

IUPAC Name:

CAPA 2043

Constituent 3

Reference substance name:

Reference substance 001

EC Number:

932-682-9

Cas Number:

31831-53-5

Constituent 4

Reference substance name:

2-Oxepanone, polymer with 1,4-butanediol

EC Number:

608-670-1

Cas Number:

31831-53-5

Molecular formula:

l(HO(CH2)5CO)-O(CH2)4O-(CO(CH2)5OH)m

IUPAC Name:

2-Oxepanone, polymer with 1,4-butanediol

Test material form:

other: liquid.

Details on test material:

- Name of test material (as cited in study report): Capa 2043
- Physical state: Colourless liquid.
- Analytical purity: 97%
- Lot/batch No.: WAC000392
- Expiration date of the lot/batch: 17th February 2014
- Storage condition of test material: Stored in the dark at room temperature.

Specific details on test material used for the study:

Identity: CAPA 2043
Label name: CAPA(TM) 2043
Chemical name: 2-oxepanone, polymer with 1,4-butanediol
Batch number: WAC000478
CAS number: 31831-53-5
EC number: 608-670-1
Expiry date: 10 May 2018
Appearance: Clear, colourless liquid
Storage conditions: Ambient controlled conditions

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Standard species and strain used for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at arrival: 27-29 days
- Weight at arrival: 78-95 g
- Fasting period before study: not specified
- Housing: The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages as indicated in the relevant SOP. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21); ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20 per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The required amount of CAPA 2043 was suspended in the vehicle. The formulation was prepared weekly at concentrations of 20, 45 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

- VEHICLE: corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in RTC Study no. A2242 in the range from 10 to 120 mg/mL. Linearity, accuracy and precision were within the limits stated in this study (r > 0.98; accuracy 80-120%; precision CV < 10%). Formulations of the test item were prepared as suspensions in corn oil. Concentration and homogeneity of the low and high dose level were assessed by taking six analytical aliquots in different positions. For the intermediate levels, only concentration was assessed by taking two different analytical aliquots. Each analytical aliquot was analysed separately. Concentration was evaluated as the mean of the single determinations and homogeneity as the coefficient of variation of the sextuplicate set.

In RTC Study no. A2242, a 3- and 8-day stability at room temperature was verified in the range from 10 to 120 mg/mL. According to this study, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (80%-120% for concentration and CV < 10% for homogeneity).

The proposed formulation procedure for the test item was checked in the range from 10 to 120 mg/mL by chemical analysis (concentration and homogeneity) in RTC Study no. A2242, to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in this study for concentration (80-120%) and homogeneity (CV <10%).

Samples of the formulations prepared on Weeks 1 and 13 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC Study no. A2242 (80-120% for concentration and CV < 10% for homogeneity).

Duration of treatment / exposure:

90 days (main groups); 90 days with 28-day recovery (satellite groups)

Frequency of treatment:

Daily for 90 days (main groups); daily for 90 days followed by a 28-day recovery period (satellite groups)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle (corn oil) control

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10/sex (main study groups); 5/sex (for recovery groups in control and high dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a preliminary non GLP compliant study (RTC Study No. E0064, see IUCLID section 7.5.1, Oberto G. (2017)). This oral gavage preliminary study was carried out at dose levels of 100, 300 and 1000 mg/kg bw/day. In this study, the test item CAPA 2043 was satisfactorily tolerated by Sprague Dawley rats at the low and intermediate dose levels (100 and 300 mg/kg bw/day), while the highest dose level of 1000 mg/kg bw/day induced some toxicity (mortality or occasional clinical signs, slight reductions in body weight and food consumption). A NOEL of 300 mg/kg bw/day was determined in this preliminary study.
As the highest dose in the preliminary study (1000 mg/kg bw/day) is not considered to be safe in a subsequent toxicity of longer duration in Sprague Dawley rats, a lower dose was chosen which is higher than the NOEL of 300 mg/kg bw/day. Thus, a high dose of 500 mg/kg bw/day was used, which is expected to induce toxicity but not death or severe suffering in a subchronic study, as required in the OECD guideline 408.
Furthermore, according to OECD guideline 408, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a NOAEL with respect to the dose selection based on OECD (NOAEL) at the lowest dose level. It is stated that two to four-fold intervals are frequently optimal for setting the descending dose levels. Since a NOAEL of 300 mg/kg bw/day was determined in the 2-week preliminary study, a mid-dose of 225 mg/kg bw was chosen for the subchronic study and a low dose of 100 mg/kg bw/day was selected based on the recommendation of the intervals of the guideline.

- Rationale for animal assignment: Control and high dose groups included 5 additional animals per sex which were sacrificed after 4 weeks of recovery. The group identification and animal numbers assigned to the treatment (males: even; females: odd) are as follows:

Group 1 (0 mg/kg bw/day) Main phase: Rat numbers: males 2-20, females 1-19; recovery phase: Rat numbers: males 22-30, females 21-29

Group 2 (100 mg/kg bw/day) Main phase: Rat numbers: males 32-50, females 31-49

Group 3 (225 mg/kg bw/day) Main phase: Rat numbers: males 52-70, females 51-69

Group 4 (500 mg/kg bw/day) Main phase: Rat numbers: males 72-90, females 71-89; recovery phase: Rat numbers: males 92-100, females 91-99


The rat numbers listed above formed the last digits of a computer generated 8 figure animal number (the remaining digits of the animal number were different for each concurrent study and served to ensure unique animal numbering for any study employing computerised data collection). The computerised system used in this study was Pristima® Version 6.4.1.

- Post-exposure recovery period in satellite groups: 4 weeks

Positive control:

Not required for this study type

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (early and afternoon)

DETAILED CLINICAL OBSERVATIONS/ NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Once before commencement of treatment and at least once per week from the start of treatment each animal was given a detailed clinical examination. Each animal was observed in an open arena
- Observations: changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern); Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions. Once during Week 12 of treatment and once during Week 4 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed. The motor activity (MA) of all animals was measured once during Week 13 of treatment and once during Week 4 of recovery by an automated activity recording. Measurements were performed using a computer generated random order.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment group, on the day before treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: weekly
- 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: No (no feeding 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: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the commencement and during Week 13 of treatment
- Dose groups that were examined: all animals in each dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: Yes, overnight food deprivation (16-17 hours)
- How many animals: 10 males and 10 females from each group
- Parameters checked in table No.1 in box “Any other information on materials & methods” were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy
- Animals fasted: Yes, overnight food deprivation (16-17 hours)
- How many animals: 10 males and 10 females from each group
- Parameters checked in table No.2 in box “Any other information on materials & methods” were examined.

URINALYSIS: No

IMMUNOLOGY: No

OTHER: Organ weights: From all animals completing the scheduled test period, the organs indicated in table 3 in box "Any other information on materials & methods incl. tables" were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3 in box in “Any other information on materials & methods incl. tables” under column “Fixation Preservation”)

HISTOPATHOLOGY: Yes (see table 3 in box “Any other information on materials & methods incl. tables” under column “Microscopic examination”)

Other examinations:

None

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male animal of the mid-dose group showed occasional signs of lethargy. This clinical sign was also observed in 6 males and 3 females of the high-dose group. Salivation was sporadically observed in 4 males of the high-dose group; slight ataxia was observed on Day 49 in one male of the mid-dose group. No clinical signs which could be considered treatment-related were observed during the recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal from the low dose group was found dead on Day 12 (Week 2) of the treatment period. Dyspnoea and piloerection were observed in this animal prior to death. At post mortem examination, this animal showed abnormal clear fluid content in the thoracic cavity and dark colour of the thymus. Chronic inflammation localized mainly in the adjacent adipose tissue and /or in the visceral serosa of the heart, lungs and thymus, considered related to uncorrect dosing, were described at histopathological examination. No treatment-induced changes were seen. All other reported microscopic observations were considered to be an expression of spontaneous and/or incidental pathology. In particular, the mild lymphocytolysis reported in the thymus can be considered stress-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant body weight and absolute body weight changes were noted between treated animals and controls during the treatment and recovery periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically significant changes were observed in food consumption during treatment and recovery periods.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Animals with no ocular abnormality were selected for the study by an ophthalmoscopic examination performed before the start of treatment. No findings were detected in both eyes of all surviving animals, from high dose and control groups, when they were re-examined during Week 13 of treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences between control and treated animals were recorded, such as increased neutrophils in mid-dose males dosed and increased haemoglobin in high dose females. Due to the slight severity or the absence of dose-relation, these findings were not considered to be of toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Fluctuations of some biochemical parameters were recorded in treated animals. In particular, glucose was increased in males dosed at 500 mg/kg/day and in all treated females (22% to 31%); calcium and inorganic phosphorus were increased in males of all treated groups (2% to 3% for calcium and 13% to 20% for phosphorus) while alkaline phosphatase was decreased in all treated females (20% to 31%). None of the above findings was dose-related. Due to the slight severity, the absence of dose-relation and/or the direction, these changes were considered of no toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes of toxicological significance were found at the weekly clinical examination, which included an evaluation of neurotoxicity. No differences between treated animals and controls, which could be considered treatment-related, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods. Motor activity measurements performed at the end of the treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Very slight reductions of terminal body weight, statistically significant in Group 3 male animals, were observed in the mid- and high dose males when compared to controls, at the end of the treatment period (-6% and -5%, respectively). Dose-related and statistically significant increases in relative weight of adrenal glands were noted in the mid- and high dose male animals (+16% and +26%, respectively), considered to be due to the slightly reduced terminal body weight in these groups in absence of other correlated changes. This change was no longer present at the end of recovery. No toxicological significance was attributed to the statistically significant increases observed in the females at the end of recovery (adrenal glands +15%, heart +15% and liver +19%), since they were not present at the end of treatment and were not supported by other relevant data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted following gross pathology examination, both in the main and recovery phases. All observed changes were considered incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted, following histopathological evaluation. All findings were considered incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

The oral toxicity of CAPA 2043 in Sprague Dawley rats, following daily oral administration at dose levels of 100, 225 and 500 mg/kg/day for 13 consecutive weeks, and recovery from any treatment-related effect during a period of 4 weeks, were investigated in this study.
One male animal, belonging to the low dose group, was found dead on Day 12 of the treatment period. Dyspnoea and piloerection were observed in this animal prior to death. The histopathological examination established that the factors contributory to the death of the animal was not related to the toxicity of the test item. Lethargy and salivation were the major signs observed (mainly in male animals) of the mid- and high dose groups, with occasional frequency, during the treatment period. These signs were reversible at the end of the recovery period. Body weight and food consumption were not affected by treatment. No other significant signs of toxic or neurotoxic effects were seen during the in vivo phase of the study. No lesions were recorded at ophthalmological examination. No changes of toxicological relevance were observed in haematological, clinical chemistry parameters and coagulation.
The relative weight of the adrenal glands was slightly increased in males treated at 225 and 500 mg/kg/day. This change was no longer observed at the end of recovery. This change was not considered to be of toxicological relevance in absence of other correlated changes (may be due to the slight reductions of terminal body weight). No other changes of toxicological relevance were observed.
No treatment-related findings were reported at post mortem macroscopic observations and at histopathological examination of animals.

In conclusion, signs of effects related to treatment with CAPA 2043 were limited to few clinical signs (lethargy, salivation and occasional ataxia) observed in animals treated at 500 mg/kg/day, when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 225 and 500 mg/kg/day. Due to the transitorial nature of these signs and to their mild severity, these signs were not considered to be adverse.
No other changes which could be considered to be adverse were observed at any of the dose levels investigated.
No treatment-related effects were observed in animals dosed at 100 mg/kg/day.

For the summary of relevant findings see table 4 in box "Any other information on results incl. tables".

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 4: Summary of relevant findings in the 90 -day study with CAPA 2043

	Males						Females
mg/kg bw/d	0	100	225	500	0R	500R	0	100	225	500	0R	500R
n	10	10	10	10	5	5	10	10	10	10	5	5
Mortality	-	1	-	-	-	-	-	-	-	-	-	-
Clinical signs	-	Piloerection, slight (1/10 animals/1.0 days per animal) Dispnoea (1/10 animals/1.0 days per animal)	Lethargy (1/10 animals/4.0 days per animal) Tooth/teeth cut (1/10 animals/5.0 days per animal) Tooth/teeth missing (1/10 animals/5.0 days per animal)	Ataxia, slight (1/15 animals/1.0 days per animal) Lethargy (6/15 animals/2.5 days per animal) Salivation (4/15 animals/1.8 days per animal	-	-	-	-	Staining, Eye, Right, Slight, Brown (1/10 animals/12.0 days per animal)	Lethargy (3/15 animals/1.0 days per animal) Tooth/teeth cut (1/15 animals/4.0 days per animal)	-	-
Weight gain (g) (1)	277.62	271.32	248.37	267.89	23.94	20.18	121.67	129.37	116.55	129.63	7.22	7.06
Haematology
Hb (g/dL)	14.86	14.94	15.27	15.10			13.68	13.60	14.04	14.18**
Neutrophils (%)	10.09	12.93*	17.11*	10.90			7.28	9.81*	8.98	7.10
Lymphocytes (%)	84.46	80.86*	77.47*	82.93			87.67	84.29*	85.73	87.38
Clinical chemistry
AP (U/L)	233.18	248.91	213.69	212.53			184.77	147.62*	127.78**	139.73**
Glucose (mg/dL)	154.79	150.34	152.66	202.39*			96.83	123.76**	123.06**	118.22*
Ca (mM)	2.638	2.719**	2.729**	2.702*			2.589	2.589	2.590	2.628
P (mg/dL)	4.894	5.522**	5.858**	5.655**			4.618	4.984	5136	5.214
Organ weights
Terminal body weight (g)	462.71	464.16	436.95**	441.63	501.74	502.32	261.70	266.60	252.66	268.64	283.66	278.74
Adrenals (g)	0.0470	0.0523	0.0515	0.0566	0.0460	0.0486	0.0565	0.0555	0.0578	0.0563	0.0558	0.0634
Adrenals (%)	0.0120	0.0113	0.0118*	0.0129*	0.0092	0.0097	0.0216	0.0209	0.0230	0.0210	0.0197	0.0227*
Heart (g)	1.4196	1.549	1.432	1.496	1.557	1.536	0.975	0.980	0.961	0.971	0.953	1.076*
Heart (%)	0.3235	0.3338	0.3276	0.3393	0.3108	0.3067	0.3279	0.3679	0.3808	0.3613	0.3371	0.3861**
Liver (g)	12.537	12.771	11.512*	12.128	17.123	16.294	6.632	7.010	6.684	6.826	7.725	9.006
Liver (%)	2.7086	2.7516	2.6336	2.7462	3.4095	3.2440	2.5330	2.6263	2.6398	2.5408	2.7159	3.2220**

R: recovery groups

*significantly different to controls (p<0.05); **p<0.01

(1) for treatment group measured on Day 91; for recovery group measured on Day 28 post-dose

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it can be concluded that the NOAEL is 500 mg/kg/day in the absence of any adverse findings of toxicological significance at any dose level. The NOEL for this study is 100 mg/kg bw/day.

Executive summary:

In a subchronic toxicity study conducted according to OECD guideline 408, the test item CAPA 2043 (2-oxepanone, polymer with 1,4-butanediol) in corn oil was administered to Sprague-Dawley rats (10/sex/dose) by gavage at dose levels of 0, 100, 225 and 500 mg/kg bw/day for 90 days. Five rats/sex were included in the high-dose and control groups for recovery assessment. The dose selection was based on a preliminary 2-week dose range finding study.

One male animal from the low dose group was found dead on Day 12, due to uncorrect dosing. Lethargy was observed in 1/10 and 6/15 male animals of the mid- and high-dose groups, respectively, and in 3/15 high-dose females. Salivation was also noted in 4/15 high-dose males of Group 4 (500 mg/kg bw/day). These signs were not observed during the recovery period. No changes of note were found at the weekly clinical examination which included an evaluation of neurotoxicity during treatment and recovery periods. No differences between treated and control groups were evident during the functional tests and at the motor activity measurements at the end of the treatment and recovery periods. Body weights and food consumption were not affected by treatment. No significant findings were detected at the ophthalmoscopic examinations at the end of the study. No changes of toxicological relevance were observed in haematological parameters. Fluctuations in some biochemical parameters were recorded in treated animals; however due to the slight severity and/or absence of a dose-response relationship, they were not considered to be of toxicological significance. Very slight reductions in terminal male body weight (significant in mid-dose males) were observed when compared to controls. No changes of toxicological relevance were observed on organ weights. Gross and microscopic pathology did not reveal any effects of treatment.

The effects of CAPA 2043 in this study were therefore limited to clinical signs (lethargy, salivation and occasional ataxia) in rats at the highest dose levels of 500 mg/kg bw/day. Due to the transient nature and mild severity, these signs were not considered to be adverse. A NOAEL of 500 mg/kg bw/day is therefore determined for this study; a NOEL of 100 mg/kg bw/day is determined.

 

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats.