Cyclopentaneacetic acid, 3-oxo-2-pentyl-, methyl ester, (1R,2S)-rel-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study at a GLP facility with no deviations. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River (Atsugi, Japan)
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 406 - 472 g; Females: 235 - 282 g
- Fasting period before study: overnight fasting of all animals prior to blood collection
- Housing: No details reported except the mating period (male: female = 1:1)
- Diet (e.g. ad libitum): NMF solid diet (Oriental yeast Co., Ltd), ad libitum
- Water (e.g. ad libitum):Mains water, ad libitum
- Acclimation period: 20 days including 3 days of quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24°C
- Humidity (%): 50 - 59%
- Air changes (per hr): 10 - 15 times/h
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark

IN-LIFE DATES: From: To:
(Main study) Males: ~ 28-d post mating; Females: ~ day 4 post partum
(Recovery group) 42-d exposure period + 2-week recovery

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: At least once every 7 days

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the preliminary validation and stability test
- Concentration in vehicle: 20, 60, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): WEN1861, WEK6144
- Purity: Not reported

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: over night - 5 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the commencement of dosing and 6-week of dosing, an aliquot of 10 mL was sampled from each concentration and the concentration was measured using GC (acetone was used as a solvent). The measured concentration for each concentration group was within 99.5 - 101.5% and achieved the acceptable range of 100 ± 10%.

GC system : HP6890N
Column : DB-5 (0.32 mm I.D. x 30 m, 0.25 μm film, Agilent Technologies Inc.)
Career gas: He
Flow: constant flow mode, 2.0 mL/min
Oven temperature program : initial 100ºC for 0 min, 280 ºC (20ºC/min for 3 min)
Injection temperature : 250 ºC
Flame ionisation detector (FID) temperature: 250 ºC
Injection volume : 1 μl

Duration of treatment / exposure:

(Main study group) Males: 42 days; Females: 41 - 46 days
(Non-mating group) 42 days

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
other: nominal conc.

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
other: nominal conc.

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
other: nominal conc.

No. of animals per sex per dose:

(Main study group) 12/sex/dose
(Non-mating group) 10/sex/control, 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on 14-day range-finding study at 250, 500, 1000 mg/kg bw/day. No effects on mortality, clinical signs or haematological examinations at any dose tested. At the early stage of dosing, a transient decrease in body weight was observed in both males and females at 1000 mg/kg bw/day, accompanied by a decrease in food consumption. An increase in cholesterol in females, an increase in the liver weights in both sexes, an increase in the kidney weights in males, as well as the protrusion in the kidneys in males were observed at 1000 mg/kg bw/day. At 500 mg/kg bw/day, a transient decrease in body weight was observed in females, accompanied by a decrease in food consumption, was observed. An increase in the liver weights was also observed in both sexes at this dose level. No effects were observed at 250 mg/kg bw/day. Therefore the dose levels for the main study was decided to be 1000, 300 and 100 mg/kg bw/day (spacing factor of 3).

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: (Dosing period) 3 times/d; (recovery period) twice/d


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: (Males and non-mating groups) once/week;
(mated females) Pre-mating: once/week; (gestation and lactation periods) GD 1, 7, 14, 20, day 4 post partum


MOTOR ACTIVITY, SENSORY REACTIVITY AND GRIP STRENGTH
(Mated females for dosing period) day 4 post partum, (the rest of animals for dosing period) the week 6 of dosing, (recovery period) day 9 of recovery period


BODY WEIGHT: Yes
- Time schedule for examinations: (mated and un-mated males) day 1, 8, 15, 22, 29, 29, 36, 42 of dosing and the day of necropsy; (recovery period) day 1, 8, 15, 22, 29, 29, 36, 42 of dosing, day 1, 8, 14 of recovery and the day of necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes
- Time schedule for examinations: (mated males and non-mated females) day 2, 8, 15, 30, 36, 42 days of dosing; (recovery period) day 2, 8, 15, 30, 36, 42 days of dosing, day 1, 8, 14 of recovery; (mated females) day 2, 8, 15 of dosing, gestation day 1, 7, 14, 20, day 2 and 4 of post partum


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for collection of blood: (main study period) In the week 6 (day 38 – 39 of dosing); (recovery group) or on 2nd week of recovery (day 10-11 of recovery).
- How many animals: (main study group) 5 animals/group; (recovery period) all animals


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After overnight withdrawal of food after the last dosing (main study groups); (recovery group) on day 14 of recovery.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 animals/group


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After overnight withdrawal of food after the last dosing (main study groups); (recovery group) on day 14 of recovery.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 animals/group

URINALYSIS: Yes
- Time schedule for collection of blood: (main study period) In the week 6 (day 38 – 39 of dosing); (recovery group) or on 2nd week of recovery (day 10-11 of recovery).
- How many animals: (main study group) 5 animals/group; (recovery period) all animals

Oestrous cyclicity (parental animals):

Yes
Daily examination of vaginal prague from the end of quarantine and grouping of animals. During dosing all the mated females were examined from the day 1 of dosing to the vaginal plug is found. For non-mated females the vaginal plug was collected from six day before necropsy to the day of necropsy

Sperm parameters (parental animals):

Parameters examined in P generation:
testes weight, epididymides weight, prostate weight, seminal vesicle weight. The macroscopic and microscopic examinations of testes, epididymides, prostate and seminal vesicle. No sperm analysis was performed.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1off-spring:
number of stillbirths and live births on day 0 postnatal, sex of pups, presence of gross anomalies, weight gain, postnatal mortality

GROSS EXAMINATION OF DEAD PUPS: Yes
external abnormalities (one offspring at 1000 mg/kg bw/day on day 0 postnatal

Postmortem examinations (parental animals):

SACRIFICE
- (main study animals) all animals, the day after the last dosing. The mated females were necropsied if 0 delivery was observed before gestation day 25; (recovery animals) all animals, after the 2-week recovery period;

ORGAN WEIGHTS: For all animals; brain, pituitary gland, thyroid gland (including parathyroid gland), adrenal gland, thymus, spleen, heart, liver, kidneys, testes, epididymides, prostate, seminal vesicle, ovaries, uterus.

GROSS NECROPSY: For all animals; brain, pituitary gland, thyroid gland (including parathyroid gland), adrenal gland, thymus, spleen, heart, liver, kidneys, testes, epididymides, prostate, seminal vesicle, ovaries, uterus.

HISTOPATHOLOGY: SFor all animals; cerebrum, cerebellum, sciatic nerve, thoracic spinal, eyes, optic nerve, Harderian gland, pituitary gland, thyroid, parathyroid gland, adrenal, thymus, spleen, submandibular lymph node, mesenteric lymph node, heart, throatic aorta, trachea, lungs (including tracheal branches), tongue, laryngeal, oesophagus, stomach, duodenum, jejunum, ileum (including Peyer's patch), cecum, colon, rectum, submandibular gland, sublingual salivary gland, liver, spleen, kidneys, urinary bladder, testes, ovaries, epididymides, uterus, vagina, prostate, seminal vesicle, skin (groin area), mammary gland (groin area), sternum (including bone marrow), femur bone (including bone marrow), skeletal muscle of femoral region, auricle

Postmortem examinations (offspring):

SACRIFICE & GROSS NECROPSY
Offspring on day 4 postnatal were subjected to external observation for gross abnormalities

Statistics:

Significant differences between Control and treated groups were expressed at the 5% (p < 0.05) or 1% (p < 0.01) level. Statistical methods used include Bartlett test, F-test, Dunnett test, Steel test, Student t-test, Aspin-Welch t-test and Fisher test.

Reproductive indices:

The mating performance and fertility parameters were calculated per group (abnormalities in oestrous cycles, mating index, fertilisation index, pregnancy index, parturition index) or per dam (gestation length, implantation index).
Abnormalities in oestrous cycles (%) = (number of animals with abnormalities in oestrous cycles /examined number of females) x 100
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100
Fertilisation index (%) = (Number of males with successful pregnancy/number of males that mated) x 100
Pregnancy Index (%) = (Number of pregnant females/Number of females mated) x 100
Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100
Gestation Length (day): Calculated as the number of days gestation including the day for observation of mating and the start of parturition
Implantation index (%) = (Number of implantation site/number of corpora lutea) x 100
Delivery index (%) = (Number of pups born/number of implantation site) x 100

Offspring viability indices:

The birth rate, stillbirth rate, external abnormalities, viability index (day 4 postnatal) and sex ratio (say 0 and 4 postnatal) were calculated per dam. The mean bodyweight of live pups were calculated per dam for each sex.
Stil birth insex (%) = (Number of stillbirth/number of pups born) x 100
External abnormality index (%) = (Number of pups with external abnormalities/number of pups born) x 100
Birth index (%) = (Number of live offspring/total number of offspring born) x 100
Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index (%) on Day 4 postnatal = (Number of live pups on Day 4/ Number of number of live pups on Day 1) x 100
Sex ratio (%) on Day 0 postnatal (including stillbirth) = (Number of male pups born/Total number of pups born) x 100
Sex ratio (%) on Day 0 postnatal (live pups) = (Number of live male pups born/Total number of live pups born) x 100
Sex ratio (%) on Day 4 postnatal = (Number of live male pups on day 4 postnatal/Total number of live pups on day 4 postnatal) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Salivation was observed in the week 3, 4, 5, 6 of dosing in 1 ∼ 2 males at 1000 mg/kg bw/day. Salivation was also observed on GD 1, 7, 14 and 20 in 1 - 5 mated female at 1000 mg/kg bw/day. Salivation was observed in the week 3, 4, 5, 6 of dosing in one non-mated female at 1000 mg/kg bw/day.
No treatment-related effects were observed at 100 or 300 mg/kg bw/day groups. No mortality or abnormal clinical signs were observed throughout the dosing and recovery periods. No treatment-related abnormalities were observed in any animals at 100 or 300 mg/kg bw/day.


OPEN FIELD OBSERVATION (PARENTAL ANIMALS):
No treatment-related abnormalities were observed in any animals at any dose levels. A statistically significant decrease in the rearing was observed in the mated females compared to the control group on lactation day 4. Since it was only marginal change and no abnormalities in rearing or detailed clinical observations were seen in the males or non-mated female animals hence it was considered incidental. No statistically significant differences were seen in the number of faeces defecated between the dosed groups and control group.


SENSORY REACTIVITY (PARENTAL ANIMALS):
(At the end of the dosing period) No abnormalities were observed in auditory response, approach response, touch response, tail pinch response, pupillary reflex or aerial righting reflex in any dosed animals. No statistical difference was observed in landing foot splay between dosed animals and control animals.

(At the end of recovery period) No abnormalities were observed in auditory response, approach response, touch response, tail pinch response, pupillary reflex or aerial righting reflex in any dosed animals. No statistical difference was observed in landing foot splay between dosed animals and control animals.


GRIP STRENGTH (PARENTAL ANIMALS):
(At the end of the dosing period) No statistical difference was observed between dosed males/non-mated females and control animals. A statistically significant increase in the hind limb was noted at 1000 mg/kg bw/day in the mated females. The changes were seen in hind limb only and no similar changes were seen in males or non-mated females. Furthermore the change was within the laboratory historical data hence it was considered not to be treatment-related. No other statistically significant change was observed.

(At the end of recovery period) A statistically significant decrease in the hind limb was observed in males at 1000 mg/kg bw/day. This was only marginal change and the effect was in hind limb only. Since no effect was seen at the end of dosing period, the observed change was considered incidental.


MOTOR ACTIVITY (PARENTAL ANIMALS):
(At the end of the dosing period) There was a statistically significant increase in motor activity during 20-30 min at 1000 mg/kg bw/day and 50 - 60 min at 100 mg/kg bw/day in males. These were either transient effect or without a dose-response relationship. No statistically significant changes were seen in the total motor activity (0-60 min). Therefore the change was considered incidental. A statistically significant decrease in motor activity was seen during 20-30 min, 30-40 min and 40-50 min intervals at 100 mg/kg bw/day and the 30-40 min interval at 1000 mg/kg bw/day in the mated females. No dose-response relationship was seen concerning the change at 100 mg/kg bw/day. The change seen at 1000 mg/kg bw/day was within the laboratory historical data. Therefore the changes seen in the mated females were considered to be incidental.

(At the end of recovery period) No statistically significant changes were seen between 1000 mg/kg bw/day group and control group.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
(Dosing period) In males, the bodyweight at 1000 mg/kg bw/day was lower than the control group throughout the dosing period with the statistically significant decrease in the bodyweight gain. A decrease in the bodyweight gain was also observed in males at 100 mg/kg bw/day but without any dose-response relationship. In the mated females a statistically significant decrease in the bodyweight gain was observed at 1000 mg/kg bw/day during gestation period.

(Recovery period) No statistically significant differences in the bodyweight gain were observed in males. A statistically significant decrease in the bodyweight gain was observed in the non-mated females at 1000 mg/kg bw/day compared to the control group.


FOOD CONSUMPTION:
(Dosing period) In males, a statistically significantly lower food consumption was observed on day 2 of dosing, followed by the higher food consumption in day 30, 36, 42 of dosing at 1000 mg/kg bw/day. A statistically significant but non-dose dependent increase in the food consumption was observed on day 8, 15 and 30 of dosing in males at 100 mg/kg bw/day. In the mated females, a statistically significant increase in the food consumption was observed on gestation day 7 and day 2 lactation at 1000 mg/kg bw/day. In the non-mated females, a statistically significant increase in the food consumption was observed on day 30 and 36 of dosing at 1000 mg/kg bw/day.

(Recovery period)
In males, a statistically significant increase in the food consumption was observed at 1000 mg/kg bw/day on day 14 of recovery period. Since this was only a slight change and no changes in the food consumption was seen on any other time point in the recovery period and as such this observation was considered to be incidental. No statistically significant differences in the food consumption were observed in the non-mated females at 1000 mg/kg bw/day.


WATER CONSUMPTION AND URINALYSIS:
(At the end of the dosing period) In males, a statistically significant increase in the water intake and urine volume was noted at 1000 mg/kg bw/day compared to the control. In the non-mated females, a statistically significant increase in the water intake, urine volume and potassium and a decrease in the osmotic pressure were noted at 1000 mg/kg bw/day compared to the control. No other abnormalities were observed at any animals tested.

(At the end of recovery period)
No other abnormalities were observed at any animals tested. In the non-mated females, a statistically significant increase in the sodium and chloride was observed at 1000 mg/kg bw/day but the change was within the laboratory historical data. Furthermore, no similar change was observed in males at the end of recovery period and as such the aforementioned changes were considered to be incidental.


HAEMATOLOGY:
(At the end of dosing period) In males, the absolute large unstained cell number (LUC) and its differential leukocyte ratio were statistically higher compared to the control at 1000 mg/kg bw/day. Since they were marginal changes and their proportions to the whole leukocytes was small, they were considered to be incidental. In the mated females, a statistically significant increase in the mean corpuscular haemoglobin concentration (MCHC) was observed at 1000 mg/kg bw/day. Since it was a only slight change and there were no other changes in the red blood cell parameters, it was considered incidental. Furthermore, a statistically significant increase in the LUC and the ratio of mono-nuclear leucocytes at 1000 mg/kg bw/day in the mated-females was only marginal changes. Since no other changes in the leukocyte parameters were seen, they were considered incidental. In the non-mated females, a statistically significant decrease in the red blood cells (RBC), haemoglobin and haematocrit was observed at 1000 mg/kg bw/day.

(At the end of recovery period) In males, a statistically significant decrease in the MCHC was observed at 1000 mg/kg bw/day. Since it was a only slight change and there were no other changes in the red blood cell parameters, it was considered incidental. A statistically significant increase in the platelet was observed at 1000 mg/kg bw/day. Since it was a only slight change and there were no other changes in the blood coagulation parameters, it was considered incidental. In the non-mated females, a statistically significant decrease in the MCHC was observed at 1000 mg/kg bw/day. Since it was a only slight change and there were no other changes in the red blood cell parameters, it was considered incidental.


CLINICAL CHEMISTRY:
(At the end of dosing period) In males, a statistically significant increase in the Blood urea nitrogen (BUN) and alkaline phosphatase (ALP) as well as a decrease in chloride in 1000 mg/kg bw/day were observed at 1000 mg/kg bw/day. A statistically significant increase in the total bilirubin was observed in males at 300 mg/kg bw/day but without any dose-response relationship. In the mated females, a statistically significant increase in the total cholesterol and BUN, as well as a decrease in the glucose were observed at 1000 mg/kg bw/day. A statistically significant increase in the gamma-glutamyl transferase (γ-GTP) at 1000 mg/kg bw/day, as well as a decrease in the total billilubin at 300 mg/kg bw/day and above, were observed. Since there were only slight changes and as such they were considered incidental. In the non-mated females, a statistically significant increase in the alanine aminotransferase (ALT), total cholesterol, triglyceride and phosphorus lipid as well as a decrease in glucose and chloride were observed at 1000 mg/kg bw/day.

(At the end of recovery period)
In males, a statistically significant decrease in lactate dehydrogenase (LDH) was observed at 1000 mg/kg bw/day. In the non-mated females, a statistically significant increase in the total protein was observed at 1000 mg/kg bw/day but this was only a marginal change.


OPHTHALMOSCOPIC EXAMINATION: Not examined.


ORGAN WEIGHTS:
(At the end of dosing period) In males, a statistically significant decrease in bodyweight was observed at necropsy. An increase in the absolute and relative liver weight and relative kidney weight was observed at 1000 mg/kg bw/day in males. A statistically significant increase in the relative liver weight was also observed at 300 mg/kg bw/day in males. A statistically significant increase in the relative brain and epididymis weights were observed at 1000 mg/kg bw/day in males although the changes were marginal with the changes in the relative weights only and therefore considered incidental. A statistically significant increase in the absolute and relative thymus weight was observed at 100 mg/kg bw/day in males although the change was not accompanied by any dose-response relationship.
In the mated females, a statistically significant increase in the absolute and relative liver weights at 300 and 1000 mg/kg bw and in the relative kidney weights at 1000 mg/kg bw/day was observed. An increase in the relative thyroid weight and decrease in the absolute uetrus weight at 1000 mg/kg bw/day were only marginal and the changes were either relative or absolute weight only and as such they were considered to be incidental. A statistically significant increase in the relative spleen and ovary weights was seen at 300 mg/kg bw/day but without any dose-response relationship.
In the non-mated females, a statistically significant decrease in the bodyweight was observed at 1000 mg/kg bw/day at necropsy. A statistically significant increase in the absolute and relative liver and kidney weights were observed at 1000 mg/kg bw/day. A statistically significant increase in the relative brain, pituitary and thyroid weights was observed at 1000 mg/kg bw/day but they were only marginal with the changes in the relative weights only. Therefore these change were considered incidental.

(At the end of recovery period) No statistically significant changes were observed in the dosed males compared to the control. A statistically significant increase in the absolute and relative liver weights was observed in females at 1000 mg/kg bw/day. Furthermore, no similar change was observed at the end of dosing period and as such that was considered to be incidental.


GROSS PATHOLOGY (PARENTAL ANIMALS):
(At the end of dosing period) In the mated females, the white focus was observed in the kidneys in 3 out of 10 animals at 1000 mg/kg bw that is considered treatment-related. No macroscopic abnormalities were observed in two mated females that did not deliver pups on GD 25. Any other macroscopic changes were considered incidental considering their occurrence and/or pathological natures.

(At the end of recovery period) Some macroscopic changes were observed but they are considered incidental considering their occurrence and/or pathological natures.


HISTOPATHOLOGY (PARENTAL ANIMALS):
(At the end of dosing period)
Liver: Minimal to mild centrilobular hepatocyte hypertrophy was observed in 5/5 males at 1000 mg/kg bw/day, 1/5 males at 300 mg/kg bw/day, 5/5 mated females at 1000 mg/kg bw/day and 5/5 non-mated females at 1000 mg/kg bw/day.
Kidneys: The minimal localised dilated renal tubules was observed in 2/7 mated females.

The mild atrophy in ovaries was observed in 2/5 non-mated females. This type of observation is rarely seen in the same strain of animals with same age but the occurence was relatively high in the observed animals. Following a track-down examination in the mated females at the lower doses, no similar change was seen. Furthermore, no similar change was seen in the mated females at 1000 mg/kg bw/day or recovery group. Therefore the aforementioned change was considered incidental.
No histopathological changes, which were considered to be associated with infertility, were observed in two mated-females at 1000 mg/kg bw/day that did not deliver pups on GD 25.
Some microscopic changes were observed but they are considered incidental considering their occurrence and/or pathological natures.

(At the end of recovery period) The minimal centrilobular hepatocyte hypertrophy was observed in 1/5 males at 1000 mg/kg bw/day.


REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
Abnormal oestrous cycle was observed in 3/12 animals although no statistical significance was noted in the count of oestrous or mean duration of oestrous cycles compared to the control.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Not examined.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
All animals mated by day 5 of cohabitation. No statistical significance was seen in the days until copulation, copulation index, insemination index or fertility index compared to the control.
A non-statistically significant decrease in the delivery index was noted at 1000 mg/kg bw/day. No statistical significance was noted in the gestation index, gestation length, number of corpora lutea, number of implantation, implantation index, number of stillborn, number of live born or external abnormalities compared to the control. A tretd in the prolonged labour time was observed in one female (animal No. 4101) at 1000 mg/kg bw/day. All females delivered pups during GD 21 - 23 and the placenta and amnions were normally treated. No abnormalities were observed in the nursing, nesting or lactation.

Dose descriptor:

NOAEL

Remarks:

Reproductive and developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects

Dose descriptor:

NOAEL

Remarks:

Reproductive and developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: P (females) and F1 (migrated information)

Dose descriptor:

NOAEL

Remarks:

Repeat dose toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING): No statistically significant difference was noted either on postnatal day 0 or 4 . All male pups from one dam died at 1000 mg/kg bw/day (no details are given in the report).


BODY WEIGHT (OFFSPRING): Bodyweight of male and female pups on postnatal day 0 was lower than control. No other statistically significant difference was seen in the bodyweight or bodyweight gain during postnatal day 0 - 4 compared to the control.


SEX RATIO (OFFSPRING): No statistically significant difference was noted either on postnatal day 0 or 4.


GROSS PATHOLOGY (OFFSPRING): No statistical significance was noted in the external abnormalities compared to the control.

Reproductive effects observed:

not specified

Conclusions:

The oral gavage administration of the test substance to rats in accordance with OECD guidance 422, at dose levels of 100, 300 and 1000 mg/kg bw/day resulted in the reduction in bodyweight gain in main group males but not in recovery group males at the end of exposure, and females and localised extension of renal tubules in females at 1000 mg/kg bw/day, as well as an increase in the liver weight in both sexes accompanied by the centrolobular hypertrophy of hepatocytes at and above 300 mg/kg bw/day. However, these changes were considered to be non adverse. An increase in the abnormalities in oestrous cycles, a trend in the prolonged labour, were not associated with poor reproductive performance. A trend in the low delivery index, as well as the lower bodyweight in male and female pups on postnatal day 1 were not significant. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for repeat dose toxicity is considered to be 1000 mg/kg bw/day. The NOAEL for reproductive and developmental toxicity for P males is 1000 mg/kg bw/day, the NOAEL for reproductive and developmental toxicity for P female as well as offspring is considered to be 1000 mg/kg bw/day.

Executive summary:

INTRODUCTION. The study was performed to assess potential adverse effects of methyl(2-pentyl-3-oxocyclopentyl)acetate on repeat dose toxicity and to screen reproductive/developmental toxicity potential. The study was conducted in accordance with the OECD Guideline for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

METHODS. The test item was administered by oral gavage to three dose groups and one concurrent vehicle control group, each of twelve male and twelve female Sprague-Dawley SPF rats (Crl:CD(SD)). Males were dosed during a two week maturation phase (42 days), pairing and up to necropsy and females were dosed during a two week maturation phase, pairing, gestation and lactation day 4 (41 – 46 days) at dose levels of 0, 100, 300 and 1000 mg/kg bw/day in corn oil. As a non-mated group, ten female rats were dosed for 42 days at the dose group of 0 and 1000 mg/kg bw/day. Following completion of dosing on day 42, five males and five mated females at 0 and 1000 mg/kg bw/day were monitored for the recovery from any treatment-related changes for 14 days.

RESULTS:

Repeat dose toxicity: No mortality. No treatment-related effects on clinical signs, sensory reactivity, grip strength or motor activity were noted.

The changes seen at 1000 mg/kg bw/day: Salivation was observed in a few males and females at 1000 mg/kg bw/day. A transient decrease in the bodyweight gain was observed in males at 1000 mg/kg bw/day, accompanied by a decrease in food consumption. A decrease in bodyweight gain was also observed in the mated females during gestation and in the non-mated females during dosing period. An increase in the food consumption was occasionally seen in males and females. An increase in water consumption and urinary volume was noted in males and non-mated females. A decrease in the osmotic pressure was noted in the non-mated females. A decrease in red blood cells, haemoglobin and haematocrit was observed in the non-mated females. In males, an increase in ALP and total cholesterol as well as a decrease in glucose were noted. In the non-mated females, a statistically significant increase in the alanine aminotransferase (ALT), total cholesterol, triglyceride and phosphorus lipid as well as a decrease in glucose were observed. An increase in liver weight and centrilobular hepatocyte hypertrophy were observed in males, mated and non-mated females. An increase in kidney weights and localised dilated renal tubules were observed in the non-mated females and mated females, respectively.

The changes seen at 300 mg/kg bw/day: An increase in liver weight and centrilobular hepatocyte hypertrophy were observed in males and mated females. A trend in recovery was seen in the aforementioned effects during 14 days of recovery period.

Reproductive/developmental toxicity: Abnormal oestrous cycle, a prolonged labour time and a trend in the decrease in delivery index were seen at 1000 mg/kg bw/day, but these were not associated with adverse effects on reproductive outcomes. Bodyweight of male and female pups on postnatal day 0 was lower than control. No treatment-related effects were observed in relation to reproductive/developmental parameters in parental animals or offspring.

CONCLUSIONS: The ‘No Observed Adverse Effect Level’ (NOAEL) for repeat dose toxicity is reported to be 1000 mg/kg bw/day. The NOAEL for reproductive and developmental toxicity for P males is 1000 mg/kg bw/day, the NOAEL for reproductive and developmental toxicity for P female as well as offspring is considered to be 1000 mg/kg bw/day.

The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study conducted according to OECD test guidelines.

Additional information

No study was available on the substance itself, therefore a read-across approach was used. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).

The results of a 90-day repeat dose toxicity study in the rat revealed no evidence to toxicity up to 100 mg/kg bw to the reproductive organs of the males or female rats used in the study. The study examined the secondary sex organs in great detail and no adverse effects were detected. In a prenatal developmental toxicity study no adverse effects were detected up to 120 mg/kg bw in the reproductive parameters and the offspring including no evidence of any change in the sex ratio.On a weight of evidence basis, the negative data of the 90-day repeat dose toxicity study and the negative data of a developmental toxicity study are together consider sufficient to obviate the need for a 2-generation reproductive study. In addition, the substance is not genotoxic.

The oral gavage administration of the test substance to rats in accordance with OECD guidance 422, at dose levels of 100, 300 and 1000 mg/kg bw/day resulted in the reduction in bodyweight gain in main group males but not in recovery group males at the end of exposure, and females and localised extension of renal tubules in females at 1000 mg/kg bw/day, as well as an increase in the liver weight in both sexes accompanied by the centrolobular hypertrophy of hepatocytes at and above 300 mg/kg bw/day. However, these changes were considered to be non-adverse. An increase in the abnormalities in oestrous cycles, a trend in the prolonged labour, were not associated with poor reproductive performance. A trend in the low delivery index, as well as the lower bodyweight in male and female pups on postnatal day 1 were not significant. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for repeat dose toxicity is considered to be 1000 mg/kg bw/day. The NOAEL for reproductive and developmental toxicity for P males is 1000 mg/kg bw/day, the NOAEL for reproductive and developmental toxicity for P female as well as offspring is considered to be 1000 mg/kg bw/day.

The registered substance is present at 10% in the supporting substance tested, i.e. well at or above the concentrations triggering classification of mixture as reproductive toxicant Category 1 or 2 as defined in the Annex VI of the Regulation (EC) No. 1272/2008, therefore it can be safely concluded that the pure cis-isomer does not need to be classified as reproductive toxicant.

Short description of key information:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening test: NOAEL Fertility = 1000 mg/kg bw/d (OECD 422, GLP, read-across, Rel. 2)

Justification for selection of Effect on fertility via oral route:
No study was available on the substance itself, therefore a read-across approach was used. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification). The key study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across,

Effects on developmental toxicity

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening test: NOAEL = 1000 mg/kg bw/d (OECD 422, GLP, read-across, Rel. 2)
Pre-natal developmental toxicity study: developmental NOAEL > 120 mg/kg bw/ day / maternal NOAEL = 80 mg/kg bw/day (OECD 414, GLP, read-across, Rel.2)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

No study was available on the substance itself, therefore a read-across approach was used. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification). The key study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across,

Additional information

No study was available on the substance itself, therefore a read-across approach was used. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).

Study No.1

An oral (gavage) developmental toxicity study in rats was performed on the supporting substance (CRL, 2007, Rel.2) according to the following FDA Guideline for Industry: Detection of toxicity to reproduction for medicinal products, ICH-S5A; September, 1994, Rockville (MD).

 

One hundred presumed pregnant Crl:CD(SD) rats were randomly assigned to four dosage groups (Groups I through IV), 25 rats per group. The test article, MethylDihydrojasmonate (MDJ), or the vehicle, Corn Oil, was administered orally via gavage once daily on days 7 through 20 of presumed gestation (DGs 7 through 20) at dosages of 0 (Vehicle), 40, 80 and 120 mg/kg/day to rats in Groups I through IV, respectively. The dosage volume (10mL/kg) was adjusted daily on the basis of the individual body weights recorded before intubation. The rats were administered the test article and/or the vehicle once daily at approximately the same time each day. All rats were examined for clinical observations of effects of the test article, abortions, premature deliveries and deaths before dosage, between one and two hours following dosage and once daily during thepost dosage period. Body weights were recorded on DG 0 and daily during the dosage and post dosage periods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21. All rats were sacrificed on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera performed. All foetuses were weighed and examined for sex and gross external alterations. Approximately half of the foetuses in each litter were examined for soft tissue alterations (by dissection) or skeletal alterations (alizarin red S-staining).

 

All prepared formulations were acceptable for use on this study.

All rats survived to scheduled sacrifice on DG 21. One dam in the 80 mg/kg/day dosage group prematurely delivered its litter on DG 21, a non dosage dependent event unrelated to MDJ.

 

The 120 mg/kg/day dosage of MDJ was associated with an increase in sparse hair coat and a significant increase in ungroomed coat that occurred at the end of the dosage period. Two rats in the 120 mg/kg/day dosage group each had tan areas in the liver lobes and a pale spleen. All other clinical and necropsy observations were considered unrelated to treatment with MDJ.

 

Toxicologically important reductions in maternal body weight gain occurred in the 120 mg/kg/day dosage group.

 

Both the 80 and 120 mg/kg/day dosage groups lost weight after the initial dosage was given (DGs 7 to 8), clear reductions in weight gain continued throughout the dosage period only in the 120 mg/kg/day dosage group. The 120 mg/kg/day dosage group had significantly reduced (p≤0.01) body weight gain on DGs 8 to 9 and DGs 7 to 10 and reduced weight gain on DGs 10 to 12. Average maternal body weights were reduced in the 120 mg/kg/day dosage group on DGs 7 through 21; these reductions were statistically significant on DGs 11 through 15, as comparison to the vehicle control group values.

 

Absolute and relative maternal feed consumption values were significantly reduced in the 120 mg/kg/day dosage group for the entire dosage period (calculated as DGs 7 to 21), as compared with the vehicle control group value. Within the dosage period, these values were reduced or significantly reduced in the 120 mg/kg/day dosage group at all tabulated intervals, as compared with the vehicle control group values, with the most severe reductions present on DGs 7 through 15.

 

No Caesarean-sectioning or litter parameters were affected by dosages of MDJ as high as 120 mg/kg/day.

Although the 120 mg/kg/day dosage group tended to have reduced foetal body weights (combined foetal body weights averaged 5.12 g, as compared with 5.24 g for the vehicle control group), the reductions in foetal body weights were not considered to be of toxicological importance because the values did not significantly differ from the vehicle control group values and were within the historical ranges of the Testing Facility.

 

No gross external, soft tissue or skeletal foetal alterations (malformations or variations) or differences in ossification site averages were attributable to dosages of MDJ as high as 120 mg/kg/day. All values were within the historical ranges of the Testing Facility.

 

Conclusions On the basis of these data, the maternal no-observable-effect-level (NOAEL) of MethylDihydrojasmonate (MDJ) is 80 mg/kg/day. The 120 mg/kg/day dosage of MDJ caused maternal weight loss after the initial dosage and reduced maternal body weight gains and body weights during the dosage period as well as reduced absolute and relative maternal feed consumption values for the entire dosage period.

 

The developmental NOAEL is greater than 120 mg/kg/day. The only observation associated with the 120 mg/kg/day dosage of MDJ was a minimal reduction in foetal body weight that was not considered to be toxicologically important because the value was not statistically significant, as compared with the vehicle control group value, and was within the historical range of the Testing Facility. Based on these data, MDJ should not be identified as a developmental toxicant.

Study No. 2

The oral gavage administration of the test substance to rats in accordance with OECD guidance 422 (Bozo 2014, Rel.2), at dose levels of 100, 300 and 1000 mg/kg bw/day resulted in the reduction in bodyweight gain in main group males but not in recovery group males at the end of exposure, and females and localised extension of renal tubules in females at 1000 mg/kg bw/day, as well as an increase in the liver weight in both sexes accompanied by the centrolobular hypertrophy of hepatocytes at and above 300 mg/kg bw/day. However, these changes were considered to be non adverse. An increase in the abnormalities in oestrous cycles, a trend in the prolonged labour, were not associated with poor reproductive performance. A trend in the low delivery index, as well as the lower bodyweight in male and female pups on postnatal day 1 were not significant. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for repeat dose toxicity is considered to be 1000 mg/kg bw/day. The NOAEL for reproductive and developmental toxicity for P males is 1000 mg/kg bw/day, the NOAEL for reproductive and developmental toxicity for P female as well as offspring is considered to be 1000 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
No adverse effects were observed in a reproductive/developmental toxicity screening test (OECD 422) at 1000 mg/kg bw/day. No adverse effects were observed in a pre natal developmental toxicity toxicty at the maximum dose level used of 120 mg/kg bw/day.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008 including ATP4.

Self-classification:

Based on the available data on a supporting substance, no additional classification is proposed.