Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 October - 30 December 2017 (in life)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 October - 30 December 2017 (in life)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague-Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are 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
- Females (nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 175-200 g (M), 151-175 g (F)
- Fasting period before study: No
- Housing: Group housing by sex prior to mating, males and females cohoused (1:1) during mating. Females house inidividually after mating; males group housed after mating.
- Diet: ad libitum
- Water: ad libitum)
- Acclimation period: approximately 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage at a dose volume of 10mL/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:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The test item was administered daily at the dose levels of 100, 300 and 1000 mg/kg bw/d using 0.5% aqueous carboxymethylcellulose as vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in the present study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations. Acceptance criteria for the validation were linearity (r >0.98), accuracy (85-115%) and precision (CV < 10%). The formulations were analysed by using HPLC method. In addition, samples of the formulations prepared during the study (the first and the last week of treatment)were analysed to check the homogeneity and concentration (acceptance criteria ± 15% of the theoretical value for concentration and CV < 10% for homogeneity). The stability of formulations at 10 and 100mg/mL was assessed at room temperature for up to 28 hours and at +4°C up to 8 days.
Duration of treatment / exposure:
Male animals were administered for two weeks before pairing, throughout the paring period and sacrificed after a total of 5 weeks of treatment. Female animals were treated for two weeks before and during the pairing, gestation and lactation periods until Day 13 post partum. Control animals were administered with the vehicle alone during the same periods.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
On the day of allocation (7 days prior to the start of treatment), all animals were weighed. Animals at the extremes of the weight distribution and those showing irregular cycle, were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects and/or contamination. Each group comprised 10 male and 10 female rats. Females were selected on the basis of pre-exposure oestrous cyclicity and animals that failed to exhibit regular cycles were not included in the groups. Males were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, for about 5 weeks. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. Females were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Positive control:
Not required for this study type.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Throughout the study, all animals were checked early in each working day in the morning and afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed approximately at the same time interval each day.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of 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, pupil size, unusual respiratory pattern). All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.


FOOD CONSUMPTION: Yes
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood samples were taken from five rats/sex and the following parameters were assesed: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelets); Prothrombin time.

CLINICAL CHEMISTRY: Yes
Blood samples were taken from five rats/sex and the following parameters were assesed: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride.

Blood samples were taken from all parental males and females for the assessment of thyroid hormones (T3, T4, TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters), 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurementswere performed using a computer generated random order. Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters), 5 males and 5 femaleswere randomly selected fromeach group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device.

IMMUNOLOGY: No

OTHER: Oestrus cyclicity
Oestrous cycles were monitored by vaginal smears in all stock females for 1 week before allocation, in order to exclude from the study females with irregular cycle (e.g. diestrous phase lasting more than 3 days). Females allocated to groups Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data were examined to determine anomalies of the oestrous cycle and pre-coital interval (i.e., the number of nights paired prior to the detection of mating). Vaginal smears were also taken from all females, before despatch to necropsy.
Sacrifice and pathology:
Gross necropsy was performed on all males and females. All females were examined also for the number of visible implantation sites (pregnant animals) and the number of corpora lutea (pregnant animals). The uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, thyroids, spleen, prostate, seminal vesicles, testes, thyroid and uterus were recorded. Histopathology was performed on tissues from rats (5/sex) of the control and high dose. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Statistics:
Standard deviations were calculated as appropriate. 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. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male in the mid-dose group was noted to have a damaged eye. This was considered to be incidental as no other clinical signs were observed during the whole duration of the study.
Mortality:
no mortality observed
Description (incidence):
All animals survived until the scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males, no effects were observed in body weight and body weight gain throughout the whole duration of the study. In females, no significant differences were noted in body weight and body weight gain during the whole duration of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by the treatment with the test item at all dose levels in both genders. Higher food consumption reported for low and mid-dose females during the pre-mating period was not consided to be adverse as there was no dose dependency.
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):
Compared with controls, males dosed at 1000 mg/kg bw/d showed a slight increase of haemoglobin (7%) and females of the same group showed decreases of platelets (9%) and reticulocytes (25%). Due to the minimal severity and/or the absence of other related findings, these changes were considered to be of no toxicological relevance. The statistically significant increase of leucocytes recorded in males dosed at 100 mg/kg/day was not dose-related, therefore it was considered incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease of phosphorus was recorded in males dosed at 100 and 300mg/kg bw/d (9% below controls). In addition, females dosed with 300 and 1000 mg/kg bw/d showed an increase of cholesterol (26% and 21%, respectively). Due to the slight severity and/or the absence of a dose-relation, the above findings were considered of no toxicological relevance. Thyroid Stimulating Hormone (TSH) was decreased in some males dosed at 1000 mg/kg bw/d. Mean group data were 52% below controls. Due to the absence of other related changes, this finding was considered of no toxicological relevance. The concentration of total Triiodothyronine (T3) and total Tyroxine (T4) did not indeed show significant differences among all groups and no correlating changes were recorded at hispathological examination of the thyroid.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effect was observed in the motor activity, sensory reactivity to stimuli and grip strength in males or females. Evaluation of the functional observation battery tests did non indicate significant differences between the test performed during the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No relevant changes were observed on terminal body, absolute and relative organ weights of treated animals that completed the treatment, when compared to the controls. The statistically significant reduction observed in absolute and relative testes weight (-8%) of high dose male group sacrificed at term was not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Animals that completed the treatment period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related. The sporadic changes such as single depressed area in kidneys of one high dose male, single or multiple dark and/or red area/s depressed or pinpoint in the glandular region of the stomach of one low dose, two mid-dose and three high dose females or small thymus in control and treated females 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:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in animals sacrificed at the end of the treatment period. The sporadic lesions such as the tubular cell degeneration in the testis of one high dose male were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The oestrous cycle evaluated before pairing was similar in all groups. One female in the low dose group and one high dose female mated during the second pairing combination. Taking this into the consideration, no test item related effects were observed.
Details on results:
There was no mortality and no treatment-related clinical signs were observed. Mean bodyweights and food consumption were unaffected by treatment. Haematology revealed minor but statistically significant reductions in platelet and reticulocyte counts in females at 1000 mg/kg bw/d. Haemoglobin concentration was slightly elevated in males of the same group. Due to the minimal severity and/or the absence of other related findings, these changes were considered to be of no toxicological relevance. There were no effects of treatment on standard clinical chemistry parameters. (TSH) was decreased in some males dosed at 1000 mg/kg bw/d. Mean group data were 52% below controls. The reduction of testes weight was of low toxicological relevance as it was not associated with any treatment-related histological findings, including no effects of the spermatogenic cycle. The concentration of total Triiodothyronine (T3) and total Thyroxine (T4) did not indeed show significant differences among all groups and no correlating changes were recorded at histopathological examination of the thyroid. FOB and motor activity assessment did not reveal any effects of treatment. Gross necropsy did not reveal any effects of treatment. Mean absolute and relative testes weights were slightly (but significantly) lower in males at 1000 mg/kg bw/d; this finding is no considered to be of toxicological significance due to the magnitude of change. There were no treatment-related histopathological findings. Oestrus cyclicity was unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
LOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

Summary of findings

 

M

F

0

100

300

1000

0

100

300

1000

Bodyweight (g) pre-mate

401.8

396.0

398.3

400.3

238.5

241.6

242.3

237.8

Bodyweight (g) terminal

407.2

395.3

406.2

404.0

 

 

 

 

Bodyweight (g) GD20

 

 

 

 

398.5

378.7

400.0

398.2

Bodyweight (g) LD13

 

 

 

 

332.6

338.2

332.4

342.9

Haematology

Hb (g/dL)

15.58

16.44

16.58

16.74*

14.86

15.05

14.38

14.96

Reticulocytes (x10e9/L)

169.5

176.5

186.8

189.0

189.0

212.2

166.4

141.6*

Reticulocytes (%)

1.91

1.90

1.98

2.00

2.42

2.66

2.24

1.80*

Clinical chemistry

Cholesterol (mg/dL)

73.50

78.58

75.64

73.98

89.12

105.64

111.94*

107.84*

T3 (ng/mL)

1.469

1.313

1.133

1.829

 

 

 

 

T4 (ng/mL)

309.9

306.3

303.7

275.7

 

 

 

 

TSH (ng/mL)

3.817

2.489

2.771

1.817**

 

 

 

 

 

 

 

 

 

 

 

 

 

Foot splay (cm)

 

 

 

 

 

 

 

 

Organ weights

Testes (g)

3.93

3.66

3.74

3.67*

 

 

 

 

Testes (%)

0.985

0.926

0.928

0.908*

 

 

 

 

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

Conclusions:
A NOAEL of 1000 mg/kg bw/d can be determined for this study in the absence of any toxicologically significant effects of treatmnet at the highest dose level of 1000 mg/kg bw/d.
Executive summary:

The repeated dose toxicity of ε-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol was investigated as part of an OECD 422 screening study. Groups of Sprague-Dawley rats were administered the test material (in aqueous carboxymethylcellulose) by gavage at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/d. Males were dosed for two weeks before pairing, throughout the paring period and sacrificed after a total of 5 weeks of treatment. Female animals were treated for two weeks before and during the pairing, gestation and lactation periods until Day 13 post partum. All animals were observed daily for mortality and clinical signs; a more detailed weekly assessment of clinical signs was performed. FOB and motor activity assessment was performed on selected rats. Blood samples were taken for the assessment of haematological and clinical chemistry parameters; thyroid hormones were also measured for males. Gross necropsy was performed on all animals; histopathology was limited to the control and high dose groups. There was no mortality and no treatment-related clinical signs were observed.  Mean bodyweights and food consumption were unaffected by treatment.  Haematology revealed minor but statistically significant reductions in platelet and reticulocyte counts in females at 1000 mg/kg bw/d.  Haemoglobin concentration was slightly elevated in males of the same group. Due to the minimal severity and/or the absence of other related findings, these changes were considered to be of no toxicological relevance.  There were no effects of treatment on standard clinical chemistry parameters.  (TSH) was decreased in some males dosed at 1000 mg/kg bw/d. Mean group data were 52% below controls. Due to the absence of other related changes, this finding was considered of no toxicological relevance. The concentration of total Triiodothyronine (T3) and total Thyroxine (T4) did not indeed show significant differences among all groups and no correlating changes were recorded at histopathological examination of the thyroid.  FOB and motor activity assessment did not reveal any effects of treatment.  Gross necropsy did not reveal any effects of treatment.  Mean absolute and relative testes weights were slightly (but significantly) lower in males at 1000 mg/kg bw/d; this finding is no considered to be of toxicological significance due to the magnitude of change.  There were no treatment-related histopathological findings.  Oestrus cyclicity was unaffected by treatment. A NOAEL of 1000 mg/kg bw/d can be determined for this study in the absence of any toxicologically significant effects of treatmnet at the highest dose level of 1000 mg/kg bw/d.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-oxydiethanol
EC Number:
203-872-2
EC Name:
2,2'-oxydiethanol
Cas Number:
111-46-6
Molecular formula:
C4H10O3
IUPAC Name:
(2-hydroxyethoxy) ethan-2-ol
Constituent 2
Chemical structure
Reference substance name:
Diethylene glycol 6-hydroxyhexanoate
Molecular formula:
C10H20O5
IUPAC Name:
Diethylene glycol 6-hydroxyhexanoate
Constituent 3
Chemical structure
Reference substance name:
Diethylene glycol di-6-hydroxyhexanoate
Molecular formula:
C16H30O7
IUPAC Name:
Diethylene glycol di-6-hydroxyhexanoate
Constituent 4
Chemical structure
Reference substance name:
Diethylene glycol tri-6-hydroxyhexanoate
Molecular formula:
C22H40O9
IUPAC Name:
Diethylene glycol tri-6-hydroxyhexanoate
Constituent 5
Chemical structure
Reference substance name:
Diethylene glycol tetra-6-hydroxyhexanoate
Molecular formula:
C28H50O11
IUPAC Name:
Diethylene glycol tetra-6-hydroxyhexanoate
Constituent 6
Chemical structure
Reference substance name:
Diethylene glycol penta-6-hydroxyhexanoat
Molecular formula:
C34H60O13
IUPAC Name:
Diethylene glycol penta-6-hydroxyhexanoat
Constituent 7
Chemical structure
Reference substance name:
Diethylene glycol hexa-6-hydroxyhexanoate
Molecular formula:
C40H70O15
IUPAC Name:
Diethylene glycol hexa-6-hydroxyhexanoate
Test material form:
liquid: viscous
Details on test material:
Appearance viscous, colorless liquid
CAS No. 36890-68-3
EINECS-No. 500-092-7

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague-Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Females (nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 175-200 g (M), 151-175 g (F)
- Fasting period before study: No
- Housing: Group housing by sex prior to mating, males and females cohoused (1:1) during mating. Females house inidividually after mating; males group housed after mating.
- Diet: ad libitum
- Water: ad libitum)
- Acclimation period: approximately 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
The test item was administered orally by gavage at a dose volume of 10mL/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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in the present study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations. Acceptance criteria for the validation were linearity (r >0.98(, accuracy (85-115%) and precision (CV < 10%). The formulations were analysed by using HPLC method. In addition, samples of the formulations prepared during the study (the first and the last week of treatment)were analysed to check the homogeneity and concentration (acceptance criteria ± 15% of the theoretical value for concentration and CV < 10% for homogeneity). The stability of formulations at 10 and 100mg/mL was assessed at room temperature for up to 28 hours and at +4°C up to 8 days.
Duration of treatment / exposure:
Male animals were administered for two weeks before pairing, throughout the paring period and sacrificed after a total of 5 weeks of treatment. Female animals were treated for two weeks before and during the pairing, gestation and lactation periods until Day 13 post partum. Control animals were administered with the vehicle alone during the same periods.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Mating was monogamous (one male to one female) with the exception indicated below. A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed. For two animals, one low dose female and one high dose female, mating with the first assigned male was unsuccessful. Therefore, the females were re-mated with a proven male of the same treatment group. The second pairing combination was monitored for mating until positive signs of copulation.
Positive control:
Not required for this study type.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Throughout the study, all animals were checked early in each working day in the morning and afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed approximately at the same time interval each day.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of 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, pupil size, unusual respiratory pattern). All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.

FOOD CONSUMPTION: Yes
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood samples were taken from five rats/sex and the following parameters were assesed: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelets); Prothrombin time.

CLINICAL CHEMISTRY: Yes
Blood samples were taken from five rats/sex and the following parameters were assesed: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride.
Blood samples were taken from all parental males for the assessment of thyroid hormones (T3, T4, TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters), 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurementswere performed using a computer generated random order. Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters), 5 males and 5 femaleswere randomly selected fromeach group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device.

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Oestrous cycles were monitored by vaginal smears in all stock females for 1 week before allocation, in order to exclude from the study females with irregular cycle (e.g. diestrous phase lasting more than 3 days). Females allocated to groups Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data were examined to determine anomalies of the oestrous cycle and pre-coital interval (i.e., the number of nights paired prior to the detection of mating). Vaginal smears were also taken from all females, before despatch to necropsy.
Sperm parameters (parental animals):
A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (4/sex where possible)

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number and sex of pups
- Stillbirths
- Llive births
- Postnatal mortality
- Weight gain
- Physical or behavioural abnormalities
- Anogenital distance (AGD)
- Presence of nipples/areolae in male pups
- Blood samples were taken for the assessment of thyroid hormones

GROSS EXAMINATION OF DEAD PUPS: Yes
Thyroid weights were recorded
Postmortem examinations (parental animals):
Gross necropsy was performed on all males and females. All females were examined also for the number of visible implantation sites (pregnant animals) and the number of corpora lutea (pregnant animals). The uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, thyroids, spleen, prostate, seminal vesicles, testes, thyroid and uterus were recorded. Histopathology was performed on tissues from rats (5/sex) of the control and high dose. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Postmortem examinations (offspring):
Gross necropsy was performed; weights of the thyroid were recorded.
Statistics:
Standard deviations were calculated as appropriate. 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. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
Fertility and copulation indices were calculated.
Offspring viability indices:
Viability was calculated on PND 0, 1, 4 and 13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male in the mid-dose group was noted to have a damaged eye. This was considered to be incidental as no other clinical signs were observed during the whole duration of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals survived until the scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males, no effects were observed in body weight and body weight gain throughout the whole duration of the study. In females, no significant differences were noted in body weight and bodyweight gain during the whole duration of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by the treatment with the test item at all dose levels in both genders. Higher food consumption reported for low and mid-dose females during the pre-mating period was not consided to be adverse as there was no dose dependency.
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):
Compared with controls, males dosed at 1000 mg/kg bw/d showed a slight increase of haemoglobin (7%) and females of the same group showed decreases of platelets (9%) and reticulocytes (25%). Due to the minimal severity and/or the absence of other related findings, these changes were considered to be of no toxicological relevance. The statistically significant increase of leucocytes recorded in males dosed at 100 mg/kg/day was not dose-related, therefore it was considered incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease of phosphorus was recorded in males dosed at 100 and 300mg/kg bw/d (9% below controls). In addition, females dosed with 300 and 1000 mg/kg bw/d showed an increase of cholesterol (26% and 21%, respectively). Due to the slight severity and/or the absence of a dose-relation, the above findings were considered of no toxicological relevance. Thyroid Stimulating Hormone (TSH) was decreased in some males dosed at 1000 mg/kg bw/d. Mean group data were 52% below controls. Due to the absence of other related changes, this finding was considered of no toxicological relevance. The concentration of total Triiodothyronine (T3) and total Thyroxine(T4) did not indeed show significant differences among all groups and no correlating changes were recorded at hispathological examination of the thyroid.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No effect was observed in the motor activity, sensory reactivity to stimuli and grip strength in males or females. Evaluation of the functional observation battery tests did non indicate significant differences between the test performed during the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in animals sacrificed at the end of the treatment period. The sporadic lesions such as the tubular cell degeneration in the testis of one high dose male were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrous cycle evaluated before pairing was similar in all groups. One female in the low dose group and one high dose female mated during the second pairing combination. Taking this into the consideration, no test item related effects were observed.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
One female in the low dose group and one high dose female mated during the second pairing combination. Taking this into the consideration, no test item related effects were observed. The outcome of mating performance (the mean time between pairing to mating and copulation index) did not show relevant differences between groups. A slightly lower fertility index was noted in the high dose group (70% compared to 90% of control group). No significant differences occurred in the number of implantation sites, incidence of preimplantation and pre-natal losses. Mean gestation length was comparable between all groups.

Details on results (P0)

There was no mortality and no treatment-related clinical signs were observed. Mean bodyweights and food consumption were unaffected by treatment. Haematology revealed minor but statistically significant reductions in platelet and reticulocyte counts in females at 1000 mg/kg bw/d. Haemoglobin concentration was slightly elevated in males of the same group. Due to the minimal severity and/or the absence of other related findings, these changes were considered to be of no toxicological relevance. There were no effects of treatment on standard clinical chemistry parameters. TSH was decreased in some males dosed at 1000 mg/kg bw/d. Mean group data were 52% below controls. Due to the absence of other related changes, this finding was considered of no toxicological relevance. The concentration of total Triiodothyronine (T3) and total Thyroxine (T4) did not indeed show significant differences among all groups and no correlating changes were recorded at histopathological examination of the thyroid. FOB and motor activity assessment did not reveal any effects of treatment. Gross necropsy did not reveal any effects of treatment. Mean absolute and relative testes weights were slightly (but significantly) lower in males at 1000 mg/kg bw/d; this finding is not considered to be of toxicological significance due to the magnitude of change. There were no treatmen-trelated histopathological findings. Oestrus cyclicity was unaffected by treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
LOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs noted in pups were pale appearance, cold to touch, small size, hair loss or black stained muzzle. The incidence of these findings did not indicate any difference between controls and treated groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test item related effects were noted in the total litter size, litter weight and mean pup weight at birth and on Days 1, 4, 14 post partum.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item related effects were noted in the total litter size, litter weight and mean pup weight at birth and on Days 1, 4, 14 post partum.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For the decedent pups, autolysis of abdominal cavity and/or absence of milk in the stomach was recorded in all groups. No abnormal findings were observed in pups sacrificed on Days 4 and 14 post partum. The only finding noted was tail tip missing for one pup in the low dose group on Day 4 post partum and for one pup in the mid-dose group on Day 14 post partum.No significant differences were detected in pup thyroidweight between controls and treated groups.
Histopathological findings:
not examined
Description (incidence and severity):
The mean anogenital distance was significantly lower in male pups of the high dose group when compared to the relative control (approximately -13%). Conversely, the mean values of the mid-dose was slightly higher but statistically significant when compared to the control group. No significant changes were seen in females. These results, even if statistically significant, were not clearly dose-related, not associated with other changes in male pups (e.g. no nipple/aerolae were recorded/retained) and therefore a clear correlation of this finding with treatment could not be established.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Pup viability and growth was comparable in all groups. There were no clinical signs related to treatment. Anogenital distance was significantly lower in the high dose male pups; however this was not clearly dose-related and was not associated with other findings (e.g. no nipples/aerolae were recorded/retained in male pups). A clear correlation of this finding with treatment could not therefore be established. Necropsy did not reveal any effects of treatment; thyroid weights were comparable in all groups.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Summary of parental effects

 

M

F

0

100

300

1000

0

100

300

1000

Bodyweight (g) pre-mate

401.8

396.0

398.3

400.3

238.5

241.6

242.3

237.8

Bodyweight (g) terminal

407.2

395.3

406.2

404.0

 

 

 

 

Bodyweight (g) GD20

 

 

 

 

398.5

378.7

400.0

398.2

Bodyweight (g) LD13

 

 

 

 

332.6

338.2

332.4

342.9

Haematology

Hb (g/dL)

15.58

16.44

16.58

16.74*

14.86

15.05

14.38

14.96

Reticulocytes (x10e9/L)

169.5

176.5

186.8

189.0

189.0

212.2

166.4

141.6*

Reticulocytes (%)

1.91

1.90

1.98

2.00

2.42

2.66

2.24

1.80*

Clinical chemistry

Cholesterol (mg/dL)

73.50

78.58

75.64

73.98

89.12

105.64

111.94*

107.84*

T3 (ng/mL)

1.469

1.313

1.133

1.829

 

 

 

 

T4 (ng/mL)

309.9

306.3

303.7

275.7

 

 

 

 

TSH (ng/mL)

3.817

2.489

2.771

1.817**

 

 

 

 

 

 

 

 

 

 

 

 

 

Foot splay (cm)

 

 

 

 

 

 

 

 

Organ weights

Testes (g)

3.93

3.66

3.74

3.67*

 

 

 

 

Testes (%)

0.985

0.926

0.928

0.908*

 

 

 

 

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

Summary of reproductive and offspring effects

 

0

100

300

1000

Oestrus cycles pre-mate (#)

2.7

2.6

2.7

2.5

Copulatory index (M)

100

90

100

90

Fertility index (M)

90

90

100

70

Copulatory index (F)

100

100

100

100

Fertility index (F)

90

100

100

70

Corpora lutea (#)

16.56

14.20

16.70

16.71

Implantations (#)

16.56

14.00

16.70

16.43

Pre-implantation loss (%)

0.00

5.53

0.00

1.56

Pre-natal loss (%)

11.03

9.35

7.20

9.83

Gestation length (d)

22.1

22.0

22.2

22.1

Litter size (#) PND0

15.56

15.75

15.90

15.29

Viable litter size (#) PND0

14.67

15.63

15.60

14.86

Litter size (#) PND1

14.67

15.38

15.20

14.71

Litter size (#) PND4

14.67

15.00

14.80

14.71

Loss (%) PND4

0.00

3.94

4.60

1.19

Litter size (#) PND13

7.78

7.88

8.00

8.00

Loss (%) PND13

0.00

1.56

0.00

0.00

Pup weight (g) PND1

6.59

6.39

6.55

6.64

Pup weight (g) PND4

9.20

8.88

9.21

9.34

Pup weight (g) PND13

30.08

29.60

29.74

30.53

AGD (M)

2.01

2.13

2.09*

1.74*

AGD (F)

1.08

1.22

1.18

1.09

T3 (ng/mL) (M)

2.013

3.845

1.159

1.870

T4 (ng/mL) (M)

110.8

121.2

85.0

117.3

TSH (M)

0.927

0.988

0.716

0.599

T3 (ng/mL) (F)

1.339

1.611

1.104

1.258

T4 (ng/mL) (F)

139.2

137.4

128.7

149.5

TSH (F)

1.244

0.983

0.930

0.751

*significantly different to controls (p<0.05)

Applicant's summary and conclusion

Conclusions:
There was no evidence in this study of adverse effects on parental rats and no evidence of treatment-related adverse effects on reproduction or offspring development. NOAELs of 1000 mg/kg bw/d for general, reproductive and developmental toxicity are therefore determined.
Executive summary:

The reproductive and developmental toxicity of ε-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol was investigated as part of an OECD 422 screening study. Groups of Sprague-Dawley rats were administered the test material (in aqueous carboxymethylcellulose) by gavage at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/d. Males were dosed for two weeks before pairing, throughout the paring period and sacrificed after a total of 5 weeks of treatment. Female animals were treated for two weeks before and during the pairing, gestation and lactation periods until Day 13 post partum. There was no mortality and no treatment-related clinical signs were observed. Mean bodyweights and food consumption were unaffected by treatment. Haematology revealed minor but statistically significant reductions in platelet and reticulocyte counts in females at 1000 mg/kg bw/d. Haemoglobin concentration was slightly elevated in males of the same group. Due to the minimal severity and/or the absence of other related findings, these changes were considered to be of no toxicological relevance. There were no effects of treatment on standard clinical chemistry parameters. TSH was decreased in some males dosed at 1000 mg/kg bw/d. Mean group data were 52% below controls. Due to the absence of other related changes, this finding was considered of no toxicological relevance. The concentration of total Triiodothyronine (T3) and total Thyroxine (T4) did not indeed show significant differences among all groups and no correlating changes were recorded at histopathological examination of the thyroid. FOB and motor activity assessment did not reveal any effects of treatment. Gross necropsy did not reveal any effects of treatment. Mean absolute and relative testes weights were slightly (but significantly) lower in males at 1000 mg/kg bw/d; this finding is not considered to be of toxicological significance due to the magnitude of change. There were no treatmen-trelated histopathological findings. Oestrus cyclicity was unaffected by treatment. One female in the low dose group and one high dose female mated during the second pairing combination. Taking this into the consideration, no test item related effects were observed.  The outcome of mating performance (the mean time between pairing to mating and copulation index) did not show relevant differences between groups. A slightly lower fertility index was noted in the high dose group (70% compared to 90% of control group).  No significant differences occurred in the number of implantation sites, incidence of preimplantation and pre-natal losses. Mean gestation length was comparable between all groups. Pup viability and growth was comparable in all groups.  There were no clinical signs related to treatment.  Anogenital distance was significantly lower in the high dose male pups; however this was not clearly dose-related and was not associated with other findings (e.g. no nipples/aerolae were recorded/retained in male pups).  A clear correlation of this finding with treatment could not therefore be established.  Necropsy did not reveal any effects of treatment; thyroid weights were comparable in all groups. There was no evidence in this study of adverse effects on parental rats and no evidence of treatment-related adverse effects on reproduction or offspring development.  NOAELs of 1000 mg/kg bw/d for general, reproductive and developmental toxicity are therefore determined.