2-Oxepanone, polymer with 1,6-hexanediol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2017 - 03 April 2018

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 November 2016 - 10 November 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 followed

Principles of method if other than guideline:

- Principle of test: The toxicity of CAPA 2047A, after daily oral administration (gavage) to rats, was investigated over a period of 2 consecutive weeks.
- Short description of test conditions: In this study, the toxicity of CAPA 2047A was tested orally on Sprague Dawley SD rats at dose levels of 100, 300 and 1000 mg/kg/day for two consecutive weeks at a dose volume of 4mL/kg body weight in corn oil.
- Parameters analysed / observed: clinical signs, mortality check, body weight and food consumption. At termination, all animals were subjected to a detailed macroscopic examination along with organ weights and tissue retention.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identity: CAPA(TM) 2047A
Chemical name: 2-oxepanone polymer with 1,6-hexanediol
Batch no.: WAD000129
CAS: 36609-29-7
EC: 609-271-5
Purity: 100% (UVCB)
Expiry: 19 July 2018
Storage: +18°C (+14-22 °C), protected from light

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.

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
- Age at arrival: 27-29 days
- Weight at arrival: males: 85-86 g and females: 90-100 g
- Housing: up to 5 of one sex per cage
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy); ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum
- Acclimation period: 18 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. 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 2047A was dissolved/suspended in the vehicle. The formulation was prepared daily at concentrations of 25, 75 and 250 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 4mL/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

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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during treatment, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day

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:
- 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.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

- OTHER: Organ weights: adrenal glands, brain (verebrum, cerebellum, medulla/pons), epididymides, heart, kidneys, liver, spleen, testes, thymus (where present)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1 under "Any other information on materials and methods incl. tables")

HISTOPATHOLOGY: No

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.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in any animal during the 2 weeks of treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed during the treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No differences were noted in body weight of male animals. The group mean body weight of high dose female rats remained lower than control (approximately -8%) for the entire treatment period. It should be noted, however, that this difference was already present at the start of dosing, and was therefore not ascribed to treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumed by treated males was similar to control animals throughout the study. A slight reduction in food consumption was noted in high dose females compared to controls, in particular during the first week of treatment. Since there was a trend of recovery over the second week of treatment, this finding was considered of minor toxicological significance.

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 changes were noted at organ weight analysis after 2 weeks of treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animals killed at termination did not show changes that could be considered treatment-related. The changes observed are suggested to be incidental or characteristically seen in untreated Sprague Dawley rats of the same age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

No death occurred and no clinical signs were observed during the study. No toxicologically relevant changes were noted in body weight since the reduction was observed at the start of dosing only. No treatment-related effects in food consumption were observed. The slight reduction in food consumption observed in the high dose females was observed in the first week of treatment and a trend od recovery over the second week of treatment indicate, that these effects are considered of minor toxicological significane. The organ weights of treated animals were similar to controls. No treatment-related changes were noted at post mortem macroscopic observations.

Key result

Dose descriptor:

other: Maximum dose level for subsequent studies

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Table 2: Summary of relevant findings in the 2 -week study with CAPA 2047A

	Males				Females
mg/kg bw/day	0	100	300	1000	0	100	300	1000
n	4	4	4	4	4	4	4	4
Mortality	-	-	-	-	-	-	-	-
Clinical signs	-	-	-	-	-	-	-	-
Mean body weight (g) Dosing Day 1/4/8/11/15	233.48/255.45/270.93/295.95/307.78	233.85/260.83/276.05/301.30/314.38	231.30/258.43/277.80/299.93/313.70	235.73/258.45/275.75/294.03/306.75	183.30/196.38/206.78/217.53/220.00	181.18/194.25/202.58/215.68/223.83	182.25/188.20/197.78/211.43/216.88	174.60/179.13/190.13*D/199.28/203.00
Mean food consumption (g) Dosing Day 8/15	19.88/20.25	20.94/20.91	21.30/21.43	20.89/20.48	15.59/16.06	14.86/15.68	14.70/14.33	12.07/14.42
Gross pathological findings
	Thymus - abnormal colour (1 animal)	Liver - abnormal shape (2 animals) Thymus - abnormal colour (2 animals)	Liver - abnormal size (1 animal)	-	-	-	-	Lungs - abnormal colour (1 animal) Thymus - abnormal area(s) (1 animal)

* Dunnett LSD Test Significant at the 0.05 level

Conclusions:

Based on the results obtained in this study, it can be concluded that no treatment-related effects were observed at any dose level, and therefore, the high dose level, 1000 mg/kg/day, could be used in a toxicity study of longer duration.

Executive summary:

In a 2-week repeated dose toxicity study, the test item CAPA 2047A (2-oxepanone polymer with 1,6-hexanediol, 100% purity(UVCB)) 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.

No death occurred and no clinical signs were observed during the study. No toxicologically relevant changes were noted in body weight and food consumption data. The organ weights of treated animals were similar to controls. No treatment-related changes were noted at post mortem macroscopic observations. Based on the results obtained in this study, it can be concluded that no treatment-related effects were observed at any dose level, and therefore, the high dose level, 1000 mg/kg/day, could be used in a toxicity study of longer duration.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

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)

Version / remarks:

Described in Council Regulation (EC) No. 440/2008 and subsequent revisions

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Oxepanone, polymer with 1,6-hexanediol

EC Number:

609-271-5

Cas Number:

36609-29-7

Molecular formula:

l(HO(CH2)5CO)-O(CH2)6O-(CO(CH2)5OH)m

IUPAC Name:

2-Oxepanone, polymer with 1,6-hexanediol

Test material form:

liquid

Specific details on test material used for the study:

Identity: CAPA(TM) 2047A
Chemical name: 2-oxepanone polymer with 1,6-hexanediol
Batch no.: WAD000129
CAS: 36609-29-7
EC: 609-271-5
Purity: 100% (UVCB)
Expiry: 19 July 2018
Storage: +18°C (+14-22 °C), protected from light

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, Italy
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at arrival: 27-29 days
- Weight at arrival: 79-101 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. 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: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20 air changes
- 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 has been 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 CAPATM 2047A was suspended in the vehicle. The formulation was prepared daily (concentrations of 25, 75 and 250 mg/mL), the day before dosing. Concentrations were calculated and expressed in terms of test item as supplied. The prepared formulations were kept under magnetic stirring overnight and up until completion of dosing.
The test item was administered orally by gavage at a dose volume of 4 mL/kg bw. 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:

Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory, according to a validated LC-MS/MS method. Analyses were carried out in a separate validation study over a range of 1-500 mg/mL. Stability was verified at room temperature for up to 8 days. Samples of the formulations prepared in Weeks 1 and 13 of the study were also analysed to check the concentration and homogeneity. The results were within the acceptance range for suspensions (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.:

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

Doses / concentrations 4

Dose / conc.:

1 000 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. E0088, see IUCLID section 7.5.1; Eileraas, 2017). This oral gavage preliminary study was carried out at dose levels of 100, 300 and 1000 mg/kg bw/day in which Sprague Dawley rats were treated by oral gavage for 2 weeks. Since no treatment-related effects were observed at any dose level, it was decided to use the same dose levels for the present study.
According to the Guideline OECD 408, a dose of 1000 mg/kg bw/day is considered to be the highest dose to be used for a limit test, except when human exposure indicates the need for a higher dose level to be used.
Furthermore, 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 408 (NOAEL) at the lowest dose level. It is stated that two to four-fold intervals are frequently optimal for setting the descending dose levels. According to this requirement, a mid-dose of 300 mg/kg bw/day and a low dose of 100 mg/kg bw/day was chosen for the dose-range finding test as well as for the main study.

- 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 are as follows (males - even numbers, females - odd numbers):
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 (300 mg/kg bw/day) Main phase: Rat numbers: males 52-70, females 51-69
Group 4 (1000 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 Weeks 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 gene rated random order.

BODY WEIGHT:
- 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): Yes
- 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

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
- 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
- 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, 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. 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):

The most notable clinical signs observed in animals of the high-dose group were ataxia in 6 out of 15 males and 1 out of 15 females, hunched posture in 2/15 females, piloerection in 3/15 females and salivation in 6/15 males. These signs were generally observed for one or only a few days during treatment. In addition, a single episode of dyspnoea was recorded in one high-dose female. No clinical signs attributable to treatment were recorded in animals of the low and mid-dose groups apart from hair loss, recorded once in a mid-dose female. No clinical signs were recorded for any animal during the recovery period.

A slight reduction in rearing was noted in treated females towards the end of treatment. The rearing was reduced in high-dose males in Week 2, while it was increased in mid- and high-dose females in Week 3. Since these findings were sporadic and not consistent, they were considered to be incidental fluctuations not related to treatment. No other differences were noted at the weekly observations of animals at removal from the cage and in an open arena. No relevant findings were found at the weekly observations of animals at removal from the cage and in an open arena during the recovery period.

Mortality:

no mortality observed

Description (incidence):

No animal died during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, a statistically significant reduction in weight gain at the high-dose was observed (12%), which is not considered toxicologically relevant. When compared to controls, a minimal decrease in terminal body weight was seen in rats of both sexes (-6% in males and -8% in females) treated at 1000 mg/kg bw/day, attaining statistical significance in females. No other changes were noted in body weight or body weight gain during the treatment and recovery periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was similar in treated and control animals throughout the study.

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. In Week 13 of treatment, all animals from control and high-dose groups were re-examined and no ocular findings were detected.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

During the dosing phase, a number of males from all treated groups showed leucocytosis, mainly due to increased lymphocytes, monocytes and basophils. Leucocytes were 47% to 57% above mean control data, with some dose-response relationship. Other statistically significant differences between control and treated animals were observed (reticulocytes in males at 1000 mg/kg bw/day 13% above controls; MCV and MCHC in females at 1000 mg/kg bw/day (4% below and 4% above controls, respectively); monocytes in females at 300 mg/kg bw/day (63% above controls). Due to the minimal severity and/or the absence of a dose-response relationship, these findings were not considered to be of toxicological relevance. A statistically significant increase of prothrombin time was recorded in animals of both sexes dosed at 1000 mg/kg bw/day (7% above controls). Due to the low severity, this change was considered of no toxicological significance.

During the recovery phase, leucocyte effects in males showed complete reversibility. The other statistically significant differences recorded between control and treated animals (decrease of mean corpuscular haemoglobin concentration and increase of reticulocytes in males and decrease of haematocrit in females) were not observed at the end of treatment, therefore they were considered to be incidental. For coagulation, no differences between control and treated animals in the recovery phase were recorded, even though data of treated animals were similar to those recorded at the end of treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Compared to controls, cholesterol was increased in males at 300 and 1000 mg/kg bw/day (12% and 15%, respectively). Glucose was increased in males at 100 mg/kg bw/day (22%) and in animals of both sexes at 1000 mg/kg bw/day (approximately 42%). The severity of these findings was not considered to be suggestive of tissue/organ injury. The statistically significant differences between control and females at 300 mg/kg bw/day (alkaline phosphatase and creatinine) were not dose-related and were therefore not considered to be related to treatment. Following the recovery phase, males at 1000 mg/kg bw/day showed cholesterol higher than controls (16%), while glucose levels showed a trend to reversibility in males and complete recovery in females.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No group differences were noted in hindlimb landing foot splay measurement in male rats, while a reduced foot splay was seen in high-dose females for one of the two recordings, which was considered to be an incidental finding. Sensory reactivity to stimuli did not show differences between groups, except for a reduction in grip strength in females at the end of treatment and in high-dose males at the end of recovery. These findings could not be clearly related to treatment, since they were not consistent between sexes and not correlated with other findings. No treatment-related differences were noted in the motor activity measurements at the end of treatment and recovery periods.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A significant decrease (~20%) in relative thymus weight was recorded for high-dose females, compared to controls. An increase (~20%) in relative adrenal weight was seen in high-dose males; however, histopathology did not reveal any effects of treatment. Minimal but statistically significant decreases (-8 or -9%) in relative spleen weight seen in males at 100 and 1000 mg/kg bw/day was considered to be incidental in the absence of a dose-response relationship or any histopathological correlates. Other statistically significant variations in mean absolute and/or relative organ weights, seen in multiple organs of treated animals, were considered related to the decrease in terminal body weight.

No organ weight effects were seen following the recovery period. The statistically significant variations in liver or kidneys weight (relative values), seen in animals previously given 1000 mg/kg bw/day, were considered incidental since all individual values laid within the physiological range of variability.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Small thymus was observed for one male and three females given at 1000 mg/kg bw/day and for two females at 300 mg/kg bw/day. No other treatment-related changes were noted following gross pathology examination in the main phase animals. All observed changes were considered spontaneous and 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.

Following recovery, small thymus was noted for two females at 1000 mg/kg bw/day. No other treatment-related changes were noted following gross pathology examination. All observed changes were considered spontaneous and 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:

effects observed, treatment-related

Description (incidence and severity):

Changes considered to be related to treatment were observed in the stomach of animals of both sexes at 1000 and 300 mg/kg bw/day; in the thymus of both sexes at 1000 mg/kg bw/day and females at 300 mg/kg bw/day. Findings in the stomach consisted of minimal to moderate hyperplasia of the squamous non-glandular epithelium, occasionally associated with submucosal edema, and were observed in the forestomach of seven males and four females at 1000 mg/kg bw/day and in three males and three females at 300 mg/kg bw/day. Minimal or mild atrophy was recorded in the thymus of four males and five females at 1000 mg/kg bw/day and in two females at 300 mg/kg bw/day. This change correlated with a decrease in relative thymus mean weight and decrease in size seen at necropsy in some treated animals. All other reported findings were considered spontaneous and 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.

After the recovery period, atrophy of the thymus was still present in one male and two females at 1000 mg/kg bw/d. Changes in the stomach had completely resolved.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

No deaths occurred, and no treatment-related findings were noted during the in-life phase of the study, including ophthalmoscopy and clinical pathology investigations. A minimal decrease was seen in terminal body weight in animals receiving 1000 mg/kg/day, statistically significant only in females. Reduced relative thymus weights were noted in high-dose females at the end of treatment. No differences in organ weights were seen at the end of the recovery period.
At macroscopic observations, small thymus was recorded at the end of treatment in few animals treated at 1000 and 300 mg/kg bw/day. Small thymus was still observed in two females previously given 1000 mg/kg bw/day at the end of the recovery period. At histopathology, minimal to moderate hyperplasia of the squamous non-glandular epithelium in the stomach was noted in several animals of both sexes treated at 1000 and 300mg/kg/day and minimal or mild thymus atrophy was observed in several animals of both sexes treated at 1000 mg/kg bw/day and in two females treated at 300 mg/kg bw/day. After the 4-week treatment-free period, atrophy of the thymus was still present in one male and two females previously dosed at 1000 mg/kg bw/day, while the changes in the stomach completely recovered. No treatment-related pathomorphological findings were observed at 100 mg/kg bw/day.

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

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Gastric histopathology seen at 300 and 1000 mg/kg bw/day represents a local (site of contact) effect and as such is not considered to be of direct relevance to the human risk assessment. Observations of small thymus and associated histopathology may represent a non-specific stress response rather than direct toxicity to this organ.

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 4: Summary of Results

	Males						Females
mg/kg bw/day	0	100	300	1000	0R	1000R	0	100	300	1000	0R	1000R
n	10	10	10	10	5	5	10	10	10	10	5	5
Mortality	-	-	-	-	-	-	-	-	-	-	-	-
Clinical signs	-	-	-	Ataxia (6/15 animals/1.5 days per animal) Salivation (6/15 animals/5.5 days per animal)	-	-	-	-	Hairloss, Muzzle (1/10 animals/1.0 days per animal)	Hunched posture (2/15 animals/1.5 days per animal) Piloerection (3/15 animals/2.7 days per animal) Ataxia (1/15 animal/1.0 days per animal) Dispnoea (1/15 animals/1.0 days per animal)	-	-
Weight gain (g) (1)	173.10	178.99	185.18	151.73*	16.12	24.21	83.11	79.14	75.34	71.71	11.88	3.74
Haematology
MCV (fL)	54.73	53.45	54.46	53.92	54.38	54.66	57.20	57.76	56.36	55.08*	56.60	55.90
MCHC (d/dL)	33.29	33.56	33.65	33.72	33.72	33.20*	33.47	33.38	34.03	34.85**	33.86	34.56
Reticulocytes (x10e9/L)	155.01	147.60	158.92	135.50*	140.76	178.60*	158.11	158.33	140.75	163.93	135.52	132.70
Reticulocytes (%)	1.866	1.778	1.905	1.646*	1.754	2.134	2.160	2.183	1.945	2.244	135.52	132.70
WBC (x10e3/µL)	4.613	6.787**	7.257***	7.181***	5.680	5.724	4.473	4.420	4.687	4.054	3.908	3.840
Lymphocytes (x10e3/µL)	3.599	5.559**	5.941**	6.140**	4.646	4.520	3.887	3.828	4.113	3.450	3.244	3.188
Basophils (x10e3/µL)	0.045	0.062	0.073*	0.070*	0.048	0.050	0.031	0.033	0.031	0.026	0.028	0.022
Neutrophils (%)	15.79	12.22*	12.34	9.41**	12.82	14.24	8.71	8.87	7..35	9.95	10.92	10.90
Lymphocytes (%)	77.85	81.52	81.81	85.45**	81.86	78.72	86.66	86.37	87.71	85.06	82.54	84.02
Monocytes (%)	2.85	3.21	3.02	2.57	2.34	3.70	1.64	1.98	2.51**	2.44**	2.78	2.32
Eosinophils (%)	2.04	1.45	1.21*	0.89*	1.76	1.84	1.77	1.41	1.13**	1.24*	2.26	1.68
Clinical chemistry
PT (s)	20.76	20.78	20.82	22.13**	23.84	23.64	22.67	21.57	22.81	24.25*	24.46	25.38
Cholesterol (mg/dL)	68.41	70.16	76.38*	78.51**	67.44	78.40**	93.50	99.67	87.42	103.17	89.48	89.50
Glucose (mg/dL)	133.07	162.33*	137.96	188.54**	187.24	172.90	118.62	110.48	122.06	166.99**	114.50	129.90
Organ weights
Terminal body weight (g)	433.30	436.68	437.95	405.39	449.68	436.38	262.85	249.20	255.51	242.67*	264.08	254.70
Spleen (g)	0.8048	0.7424	0.8118	0.6830**	0.8250	0.7902	0.5866	0.5589	0.5855	0.5320	0.5630	0.5322
Spleen (%)	0.1856	0.1701*	0.1857	0.1683*	0.1835	0.1809	0.2224	0.2249	0.2296	0.2196	0.2133	0.2095
Thymus (g)	0.3530	0.3514	0.3624	0.2859	0.3106	0.2650	0.2854	0.2535	0.2456	0.2119**	0.2646	0.2328
Thymus (%)	0.0809	0.0799	0.0828	0.0704	0.0694	0.0608	0.1086	0.1020	0.0958	0.0875**	0.1005	0.0904
Pathology
Thymus: small	-	-	-	1/10	-	-	-	-	2/10	3/10	-	2/5
Stomach: squamous hyperplasia	-	-	-	7*10	-	-	-	-	3/10	4/10	-	-
Thymus: atrophy	-	-	-	4/10	-	1/5	-	-	2/10	5/10	-	2/5

*significantly different to controls (p<0.05); **p<0.01; ***p<0.001

(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 100 mg/kg/day based on the histopathology of the thymus and stomach at 300 mg/kg bw/day.

Executive summary:

In a subchronic toxicity study conducted according to OECD guideline 408, the test item CAPA 2047A (2-oxepanone polymer with 1,6-hexanediol) in corn oil was administered to Sprague-Dawley rats (10/sex/dose) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day for 90 days. Five rats/sex were included in the high-dose and control groups for 4-week recovery assessment. The dose selection was based on a preliminary 2-week dose range finding study.

 

No deaths occurred during the study period. Clinical signs were observed in animals of the high-dose group, including ataxia in 6/15 males and 1/15 females, hunched posture in 2/15 females, piloerection in 3/15 females and salivation in 6/15 males. These signs were generally observed for one or only a few days during treatment.  A single episode of dyspnoea was recorded in one high-dose female. A minimal decrease in terminal body weight was seen in rats of both sexes treated at 1000 mg/kg bw/day, attaining statistical significance in females. Overall weight gain was lower in males and females in the high-dose group; significantly in males. Food consumption was comparable in all groups. Ophthalmoscopy did not reveal any effects of treatment. Clinical pathology investigations did not reveal any effects of treatment considered to be of toxicological significance. Reduced relative thymus weights were noted in high-dose females at the end of treatment. No differences in organ weights were seen at the end of the recovery period. Gross necropsy revealed small thymus at the end of treatment in a small number of rats at 1000 and 300 mg/kg bw/day; small thymus was also observed in two females at 1000 mg/kg bw/day at the end of the recovery period. Histopathology revealed minimal to moderate hyperplasia of the squamous non-glandular epithelium of the stomach in several animals of both sexes at 1000 and 300 mg/kg bw/day and minimal or mild thymus atrophy was observed in several rats of both sexes treated at 1000 mg/kg bw/day and in two females at 300 mg/kg bw/day. After the 4-week recovery period, atrophy of the thymus was observed in one male and two females previously at 1000 mg/kg bw/day, while the changes in the stomach had completely resolved. No histopathological effects of treatment were observed at 100 mg/kg bw/day. Gastric histopathology seen at 300 and 1000 mg/kg bw/day represents a local (site of contact) effect and as such is not considered to be of direct relevance to the human risk assessment. Observations of small thymus and associated histopathology may represent a non-specific stress response rather than direct toxicity to this organ but are taken into account for derivation of the study NOAEL. A NOAEL of 100 mg/kg bw/day is therefore determined for this study.