(2E)-2-ethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)but-2-en-1-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a recent GLP combined repeated dose and reproduction / developmental screening test conducted according to OECD guideline 422, no impact of the test item was identified on fertility of rats exposed by gavage up to 1000 mg/kg bw/day for 2 weeks before mating. Also, in a recent GLP 90-day repeated dose toxcity study, no adverse effects, especially in reproductive organs, were observed in rats exposed by diet up to 981 mg/kg bw/day for males and 1109 mg/kg bw/day for females.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2010 to 17 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): CAS 28219-61-6, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2 buten-1-ol
- Physical state: liquid
- Analytical purity: 98.9%
- Lot/batch No.: 094678
- Storage condition of test material: at ambient temperature (approximately 20°C), protected from light

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 301 to 376 g for males and 198 to 260 g for females
- Housing: up to 5 during pre-mating for all animals and after mating for males and during toxicity phase for unmatted females, individually with litter for females during gestation and lactation.
- Diet (e.g. ad libitum): standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Main phase males, Toxicity phase females and Recovery phase animals.
- Water (e.g. ad libitum): potable water taken from the public supply, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 April 2010 To: 25 June 2010

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: approximately 50% of the final volume of corn oil was added to the required amount of test material. The formulation was mixed using a magnetic stirrer until all of the test material had dissolved and more corn oil was added to make up the required volume. The formulation was then mixed using a magnetic stirrer until homogeneous.
Initially all formulations were prepared freshly on the day of use and used within two hours of completion of preparation. However, following confirmation of the results from a homogeneity and stability, formulations were prepared weekly, subdivided into daily aliquots and used within 8 days of preparation.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last weeks of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two assays from each group were analysed. The mean concentrations of CAS 28219-61-6 in test formulations analysed for the study were within applied limits, +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. An additional five males and five females were dosed with the vehicle or at 1000 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose Offspring were not dosed.

Frequency of treatment:

Once a day, 7 days a week

Details on study schedule:

None

Dose / conc.:

100 ppm

Remarks:

actual ingested

Dose / conc.:

300 ppm

Remarks:

actual ingested

Dose / conc.:

1 000 ppm

Remarks:

actual ingested

No. of animals per sex per dose:

Toxicity subgroup: 10 males and 5 females/dose (except for control males and at top dose: 5 males/dose)
Reproductive subgroup: 10 females/dose and same males as for toxicity subgroup
Recovery subgroup: 5 males and 5 females /dose (control and top dose); Recovery phase males also used for pairing with Main reproductive phase females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in a preliminary study (Huntingdon Life Sciences Study Number: OAD0001), dose levels of 150, 600 and 1000 mg/kg bw/day were employed and treatment at dose levels up to 1000 mg/kg/day was generally well tolerated. In that study animals dosed at 600 and 1000 mg/kg/day showed post dose observations of salivation and chin rubbing and at 1000 mg/kg/day signs included slightly low weight gain in males, initially lower food consumption in females, increased water consumption and at necropsy increased liver weights. The dose levels selected for the study included a high dose of 1000 mg/kg/day as the limit dose specified in the OECD 422 guideline and may generate some toxic reactions, the low and intermediate dose levels are selected to establish a no observed adverse effect level to give suitable safety margins and establish dose response relationships.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Animals and cages were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded in relation to dose administration. For the Main phase males and Toxicity phase females dosing observcations were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For Main phase females these were recorded daily during the first week of dosing, twice weekly during Week 2 of dosing, on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation. Observations were recorded at the following times in relation to dose administration:
Pre-dose
On return of the animal to its home cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal (physical condition and behaviour during handling with particular attention to possible signs of neurotoxicity). For the Reproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.A weekly physical examination including arena observations was performed during the recovery period.

BODY WEIGHT:
The weight of the Main phase males and Toxicity phase females was recorded on the day that dosing commenced (Week 0), weekly throughout the dosing and recovery periods and before necropsy. Main phase females were weighed on the day that dosing commenced (Week 0), weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the start of study for Main phase males and Toxicity phase females and Main phase females until the animals were paired for mating. Food consumption was recorded weekly (g/animal/week) during the recovery period. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage. Food consumption was not recorded for Main phase males and females during pairing.
For each Main phase female after mating, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-6, 7-13 and 14-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation.

OTHER:
- SENSORY REACTIVITY:
Sensory reactivity and grip strength assessments were performed (before dosing) on the second 5 main phase (group 2 and 3)/recovery group (control and group 4) males and on toxicity phase (Groups 2 and 3)/recovery phase (Control and Group 4) females during Week 5 of study. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle reflex, tail pinch response and grip strength.

- MOTOR ACTIVITY:
During Week 5 of study (before dosing), the motor activity of the second five main phase (Groups 2 and 3)/recovery phase (Control and Group 4) males and on toxicity phase (Groups 2 and 3)/recovery phase (Control and Group 4) females was measured using a Rodent Activity Monitoring System (Version 2.0.3). Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).

- HAEMATOLOGY:
During Week 5 of treatment (before dosing on each occasion)and after 2 weeks of recovery , blood samples were obtained from the first five main phase males and on the toxicity phase females after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein. The following were measured using a Bayer Advia 120 haematology analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)) and Platelet count (Plt). The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded. Prothrombin time (PT) (using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent) and Activated partial thromboplastin time (APTT) (using an ACL 3000 Plus Analyser and IL APTT reagent) were also measured.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected and the plasma was examined using a Roche P Modular Analyser for: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile Acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb) (by chemical assay).

Oestrous cyclicity (parental animals):

For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from Main phase females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

There were no changes in the reproductive organs which were considered test article related.
The seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Litter observations:

PARAMETERS EXAMINED:
All litters were examined at approximately 24 hours after birth and then daily thereafter for clinical signs (evidence of ill health or reaction to treatment), litter size (mortality and consequent changes in litter size from Days 1-7 of age), sex ratio of each litter (recorded on Days 1, 4 and 7 of age) and individual bodyweight (recorded on Days 1, 4 and 7 of age).

GROSS EXAMINATION OF PUPS:
All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined.

Postmortem examinations (parental animals):

SACRIFICE:
Toxicity subgroup males and females and Reproductive subgroup males were killed in Week 6 after completion of the Week 5 investigations.
Reproductive subgroup females were killed on Day 7 of lactation.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For Reproductive subgroup females, the number of uterine implantation sites was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 1 were weighed.
In the Toxicity subgroup, the following organs were fixed for histopathology: Adrenal glands, Peyer’s patch, Brain, Pituitary, Caecum, Prostate, Colon, Rectum, Duodenum, Sciatic nerves, Epididymides (L&R), Seminal vesicles and coagulation gland, Heart, Spinal cord, Ileum, Spleen, Jejunum, Sternum with marrow, Kidneys, Stomach, Liver, Testes (L&R), Lungs, Thymus, Lymph nodes (left axillary and mesenteric), Thyroid with parathyroids, Trachea, Mammary area (caudal), Urinary bladder, Oesophagus, Uterus with cervix and oviducts, Ovaries (L&R) and Vagina.
In the Reproductive subgroup, the following organs were fixed for histopathology: Mammary area (caudal), Testes (L&R), Ovaries (L&R), Uterus with cervix and oviducts, Pituitary, Vagina and Prostate. Samples of any abnormal tissues were also retained and processed for examination.
All tissues preserved for examination were examined for all Toxicity subgroup and Reproductive subgroup animals of Control group and 900 mg/kg bw/day group. Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals.

Postmortem examinations (offspring):

All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were also examined.

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, litter size and survival indices and clinical pathology data: 1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. 2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.
For gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.

Reproductive indices:

Percentage mating : Number animals mating / Animals paired × 100
Conception rate (%) : Number animals achieving pregnancy / Animals mated × 100
Fertility index (%) : Number animals achieving pregnancy / Animals pairing × 100

Offspring viability indices:

Gestation index (%) : Number of live litters born / Number pregnant × 100
Post - implantation survival index (%) : Total number offspring born / Total number uterine implantation sites × 100
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Lactation index (%) : Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering × 100
Percentage of males : Number of males in litter/ Total number of offspring in litter x100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day: Three females were killed for humane reasons on Day 21 or 22 of gestation because of general loss of condition. Underactive behaviour in males and females and unsteady posture in females during Week 1: resolved within the working day

Mortality:

mortality observed, treatment-related

Description (incidence):

Three animals (No’s 47, 48 and 50) from the high dose group (1000 mg/kg/day) were killed for humane reasons on Day 21 or 22 of gestation because of general loss of condition. The female on Day 22 of gestation (No.50) had started parturition.Typical terminal signs included pale, dull or partially closed eyes, irregular, deep or shallow breathing, reduced activity, hunched posture, piloerection, and in the animal that had started parturition, reduced body tone and limited use of hind limbs. Females killed on Day 21 of gestation had live litters but those born on Day 22 of gestation died soon after birth. The incidence of resorptions was not remarkable and there were no major maternal findings at necropsy.
One female (No.46) was killed on Day 1 of lactation due to high levels of pup mortality: many of the pups died soon after birth and live pups were small (none heavier than 4.6g), cold to touch and had no milk visible in the stomach. The macroscopic examination of the dam revealed only a pale area on the visceral surface of the left liver lobe. Most of the tissues microscopically examined were unremarkable and the changes identified in the reproductive organs were all within the normal physiological changes post parturition.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day: males showed lower overall weight gain (Week 0-5) compared with Control. Lower bodyweight gain of females during late gestation (Days 14-20) resulting in lower overall (Day 0-20) bodyweight gain compared with Control .

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day: males showed lower overall weight gain (Week 0-5) compared with Control. Lower bodyweight gain of females during late gestation (Days 14-20) resulting in lower overall (Day 0-20) bodyweight gain compared with Control .
Females receiving 1000 mg/kg/day showed lower food consumption during late gestation (Days 14-19) corresponding with the period of lower body weight gain.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Visual assessment of water consumption indicated that males and females receiving 1000 mg/kg/day were consuming more water than the Controls during the dosing period.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no marked effects of CAS 28219-61-6 upon haematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

During week 5 of dosing, alanine aminophosphotase and alanine aminotransferase and cholesterol levels were statistically higher than Controls in females receiving 1000 mg/kg/day with an indication of a slightly increase levels of alanine aminophosphotase for females receiving 300 mg/kg/day. Increased bilirubin plasma levels also attained statistical significance. No similar effects were seen for males. Males receiving CAS 28219-61-6 at dose levels of 1000 mg/kg/day had slightly lower calcium levels by Week 2 of recovery, calcium levels amongst these males were still low when compared with Controls. These limited effects were considered as adaptative and/or of no toxicological relevance. All other previously affected parameters were essentially similar to those of the Controls, thus indicating complete recovery.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centrilobular hepatocyte hypertrophy was seen in the liver of two toxicity phase females receiving 1000 mg/kg/day. This finding is considered an adaptive change due to the test article and is accountable for the variation in liver weight and the enlarged appearance of the liver as reported in some animals at necropsy. The microscopic examination for the liver of the toxicity phase females receiving 100 and 300 mg/kg/day and for the females dosed at 1000 mg/kg/day but sacrificed after 2 weeks recovery period did not reveal the presence of centrilobular hepatocyte hypertrophy and confirmed that a recovery from this liver change had taken place.
No findings were seen in the adrenal glands to account for the pale appearance reported at necropsy.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Females receiving 1000 mg/kg/day showed lower food consumption during late gestation (Days 14-19) corresponding with the period of lower body weight gain.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Administration of CAS 28219-61-6 had no effect on oestrous cycles.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

prematuretermination of three out of ten pregnant females dosed at 1000 mg/kg/day due to the loss of clinical condition in late gestation/during parturition (after approximately 5 weeks of dosing).

MORTALITY (PARENTAL ANIMALS):
Three animals (No’s 47, 48 and 50) from the high dose group (1000 mg/kg/day) were killed for humane reasons on Day 21 or 22 of gestation because of general loss of condition. The female on Day 22 of gestation (No.50) had started parturition.Typical terminal signs included pale, dull or partially closed eyes, irregular, deep or shallow breathing, reduced activity, hunched posture, piloerection, and in the animal that had started parturition, reduced body tone and limited use of hind limbs. Females killed on Day 21 of gestation had live litters but those born on Day 22 of gestation died soon after birth. The incidence of resorptions was not remarkable and there were no major maternal findings at necropsy.
One female (No.46) was killed on Day 1 of lactation due to high levels of pup mortality: many of the pups died soon after birth and live pups were small (none heavier than 4.6g), cold to touch and had no milk visible in the stomach. The macroscopic examination of the dam revealed only a pale area on the visceral surface of the left liver lobe. Most of the tissues microscopically examined were unremarkable and the changes identified in the reproductive organs were all within the normal physiological changes post parturition.

CLINICAL SIGNS (PARENTAL ANIMALS):
Underactive behaviour in males and females and unsteady posture in females were observed during Week 1 in animals at 1000 mg/kg/day: these signs resolved within the working day. Salivation/post salivation staining and/or chin rubbing have been observed post-dosing in all
dose groups, with the incidence of these findings increased with increasing dose level. These signs are often observed following oral administration of substances with a low palatability and are not considered to be an adverse toxic effect.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
There were no clear effects on bodyweight in males receiving up to 1000 mg/kg/day. Weight gains were variable and without a clear dose response. Males receiving 1000 mg/kg/day showed significantly lower overall (week 0-5) weight gain (78% of control) with the major effects on weight gain during weeks 2-3 and 4-5.
Bodyweight during the recovery phase was similar to controls.
During late gestation (Days 14-20) bodyweight gain of females receiving 1000 mg/kg/day was significantly lower than Control, this resulted in lower overall (Day 0-20) bodyweight gain. Bodyweight and bodyweight gain were unaffected during lactation.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
There was no clear effect on food consumption in the males or unmated females.
During late gestation (Days 14-19) food consumption lower than Control was recorded for females receiving 1000 mg/kg/day, corresponding with the period of low bodyweight gain. There was also a suggestion of lower food consumption during lactation but this did not
attain statistical significance.

WATER CONSUMPTION:
Visual assessment of water consumption indicated that males and females receiving 1000 mg/kg/day were consuming more water than the Controls during the dosing period.

REPRODUCTIVE FUNCTION (ESTROUS CYCLE) AND REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
There was no effect of CAS 28219-61-6 on oestrous cycles, precoital interval, mating performance or fertility. All females mated and were pregnant but at 1000 mg/kg/day there was evidence of increased sensitivity in late gestation: two females had to be terminated before giving birth and one female was terminated during parturition, leading to a reduction in the gestation index. Gestation length was within normal range but there was a slight
increase in the numbers of animals having longer (23 day) gestation periods. All animals in the Control, 100 and 300 mg/kg/day groups gave birth to a live litter.
There was no effect of test material on the number of implantations but at 1000 mg/kg/day post implantation survival index, live birth index and viability index were all lower than Control so that live litter size was smaller. Lactation index assessed on Day 7 of lactation was unaffected. There was no effect at dose levels of 300 mg/kg/day or below. Male and female offspring bodyweights were not adversely affected by CAS 28219-61-6.

ORGAN WEIGHTS (PARENTAL ANIMALS):
The analysis of unadjusted and statistically adjusted organ weights for Toxicity subgroup animals indicated high liver weights in males and females receiving 300 and 1000 mg/kg/day at 112/128% of male control and 122/163% of female control values respectively. Kidney weights in females receiving 1000 mg/kg/day were above Control (124% of control). After two weeks of recovery the intergroup differences in organ weights were no longer apparent.

GROSS PATHOLOGY (PARENTAL ANIMALS):
Enlarged livers were observed in toxicity subgroup females receiving 1000 mg/kg/day o. Pale adrenals were observed in females receiving 300 and 1000 mg/kg/day and a dose response was apparent. There were no significant necropsy findings for females on Day 7 of lactation.

HISTOPATHOLOGY (PARENTAL ANIMALS):
Centrilobular hepatocyte hypertrophy was seen in the liver of two toxicity phase females receiving 1000 mg/kg/day . This finding is considered an adaptive change due to the test article and is accountable for the variation in liver weight and the enlarged appearance of the liver as reported in some animals at necropsy. The microscopic examination for the liver of the toxicity phase females receiving 100 and 300 mg/kg/day and for the females dosed at 1000 mg/kg/day but sacrificed after 2 weeks recovery period did not reveal the presence of centrilobular hepatocyte hypertrophy and confirmed that a recovery from this liver change had taken place.
No findings were seen in the adrenal glands to account for the pale appearance reported at necropsy.


OTHER FINDINGS (PARENTAL ANIMALS):
SIGNS AND ARENA OBSERVATIONS:
There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment.

SENSORY REACTIVITY OBSERVATIONS AND GRIP STRENGTH:
Sensory reactivity observations and grip strength values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.

MOTOR ACTIVITY:
Motor activity scores for Toxicity subgroup males and females were considered to be unaffected by treatment.

HAEMATOLOGY:
There were no marked effects of CAS 28219-61-6 upon haematology parameters.

BLOOD CHEMISTRY:
During week 5 of dosing, alanine aminophosphotase and alanine aminotransferase and cholesterol levels were statistically higher than Controls in females receiving 1000 mg/kg/day with an indication of a slightly increase levels of alanine aminophosphotase for females receiving 300 mg/kg/day. Increased bilirubin plasma levels also attained statistical significance. No similar effects were seen for males. Males receiving CAS 28219-61-6 at dose levels of 1000 mg/kg/day had slightly lower calcium levels by Week 2 of recovery, calcium levels amongst these males were still low when compared with Controls. All other previously affected parameters were essentially similar to those of the Controls, thus
indicating complete recovery.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

mated female

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

unmated female

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

other: only adaptative effects on liver

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs of offspring did not indicate any reaction to maternal exposure to CAS 28219-61-6.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Post implantation survival index, live birth index and viability index for animals receiving 1000 mg/kg/day were all slightly lower than Control but intergroup differences were not statistically significant.

Body weight and weight changes:

no effects observed

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:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

LITTER SIZE, SEX RATIO AND SURVIVAL INDICES:
There was no effect of test material in the number of implantations in all dose groups. At 1000 mg/kg/day, total litter size on Day 1 was marginally lower than control, and live litter size throughout was significantly smaller for animals receiving 1000 mg/kg/day. The apparently smaller litter size in the animals receiving 300 mg/kg/day is primarily attributable to the lower number of implantations for this group and is considered to be part of natural variation consequent to the small group size. There was no effect at 300 mg/kg/day.
Post implantation survival index, live birth index and viability index for animals receiving 1000 mg/kg/day were all slightly lower than Control but intergroup differences were not statistically significant. Lactation index assessed on Days 7 of lactation was unaffected. The litters of females (no.47, 48 and 50) have been excluded from the survival index and sex ratio as they did not complete successful parturition and the death of the dam directly attributable to the death of the litter. There was no effect at dose levels of 300 mg/kg/day or below.
Sex ratio was unaffected by administration of CAS 28219-61-6 up to dose levels of 1000 mg/kg/day.

CLINICAL SIGNS (OFFSPRING): There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.

BODY WEIGHT (OFFSPRING): .Male and female offspring bodyweights were considered to be unaffected by CAS 28219-61-6.

GROSS PATHOLOGY (OFFSPRING): Necropsy findings of offspring killed at the end of the study or killed or dying prior to scheduled termination did not indicate any reaction to maternal dosing with CAS 28219-61-6.

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

no

Relevant for humans:

not specified

Body weight change in Main Phase males:

Group		Change 0-1	%	Change 1-2	%	Change 2-3	%	Change 3-4	%	Change 4-5	%	Change 0-5	%	R1-R2
control	Mean SD N	37 5.7 10		45 6.0 10		20 5.6 10		29 11.0 10		21 6.1 10		153 26.8 10		12 9.1 5
100 mg/kg/d	Mean SD N	33 9.1 10	87	35 10.3 10	78	21 5.8 10	101	20 10.0 10	70	24 5.6 10	111	132 26.3 10	87
300 mg/kg/d	Mean SD N	38 9.5 10	101	41 6.4 10	90	26 6.4 10	130	29 10.5 10	101	20 5.6 10	96	154 28.5 10	101
1000 mg/kg/d	Mean SD N	33 9.6 10	87	38 8.8 10	84	13 6.8 10	66	22 9.0 10	77	13 10.5 10	63	119 31.5 10	78	13 8.3 5

Blood chemistry in Toxicity Subgroup females:

Group		ALP U/L	ALT U/L	Bili µmol/L	BIAC µmol/L	Creat µmol/L	Chol mmol/L
control	Mean SD N	71 14.8 5	40 7.1 5	2 0.4 5	14.2 4.01 5	28 2.1 5	1.51 0.414 5
100 mg/kg/d	Mean SD N	79 15.6 5	36 5.6 5	2 0.0 5	20.4 8.84 5	40** 5.1 5	1.35 0.236 5
300 mg/kg/d	Mean SD N	101 36.4 5	40 16.1 5	2 0.4 5	22.8 10.02 5	33 2.7 5	1.72 0.200 5
1000 mg/kg/d	Mean SD N	137** 32.1 5	70** 9.5 5	3** 0.4 5	29.5* 11.84 5	37** 3.6 5	2.49** 0.216 5

BIAC: Bile Acids; Bili: Total Bilirubin.

Blood chemistry in toxicity subgroup females - Recovery Week 2:

Group		ALP U/L	ALT U/L	Bili µmol/L	BIAC µmol/L	Creat µmol/L	Chol mmol/L
control	Mean SD N	65 17.9 5	39 6.6 5	2 0.4 5	32.3 18.29 5	7.17 0.447 5	2.06 0.377 5
1000 mg/kg/d	Mean SD N	62 13.2 5	37 14.5 5	2 0.0 5	50.4 47.42 5	7.54 1.167 5	2.33 0.230 5

Liver weight in males:

Group		Toxicity Phase			Recovery Phase
		Terminal body weight	Liver unadjusted	Liver ajusted	Terminal body weight	Liver unadjusted	Liver ajusted
control	Mean SD N	487 43 5	20.627 1.135 5	19.996	515 31 5	23.321 2.798 5	23.534
100 mg/kg/d	Mean SD N	469 34 10	20.048 2.106 10	20.287
300 mg/kg/d	Mean SD N	496 45 10	23.493 3.295 10	22.396**
1000 mg/kg/d	Mean SD N	427** 33 5	23.341 1.863 5	25.686**	522 42 5	21.833 2.538 5	21.620

Liver weight in females:

Group		Toxicity Phase			Recovery Phase
		Terminal body weight	Liver unadjusted	Liver ajusted	Terminal body weight	Liver unadjusted	Liver ajusted
control	Mean SD N	277 25 5	10.293 1.153 5	10.456	282 22 5	11.638 1.268 5	11.846
100 mg/kg/d	Mean SD N	298 24 5	11.601 1.027 5	11.251
300 mg/kg/d	Mean SD N	274 14 5	12.489 1.091 5	12.738**
1000 mg/kg/d	Mean SD N	286 18 5	17.079 0.941 5	17.016**	294 24 5	12.835 1.768 5	12.627

Conclusions:

The No-Observed-Adverse–Effect-Level (NOAEL) for males was 300 mg/kg/day, the NOAEL for maternal toxicity was 300 mg/kg/day and the NOAEL for reproduction/developmental toxicity was at least 300 mg/kg/day. The NOAEL for unmated females was 1000 mg/kg bw/day.

Executive summary:

In a GLP study conducted according to OECD guideline 422, three groups, each comprising of ten male and ten female rats for the Main (reproductive) phase and five female rats for the Toxicity phase received the test item at doses of 100, 300 or 1000 mg/kg/day at a dose volume of 5 mL/kg/day. Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. An additional five males and five females were dosed with the vehicle or at 1000 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

In the 1000 mg/kg/day dose group, four females allocated to the littering phase of this study were killed prior to scheduled termination. Two females were killed in late gestation following deterioration in clinical condition. The females showed signs including irregular breathing, reduced activity, hunched posture and dull/pale/partially closed eyes. Both females were pregnant with a live and normal litter. A further female was killed during parturition with similar signs, plus limited use of limbs and reduced body tone: most pups had died and the three that remained were killed at the same time as the dam. The fourth female was killed on Day 1 of lactation due to high levels of pup mortality on Day 1 of lactation. The condition of the litter was poor with small, cold pups that did not appear to be feeding. The macroscopic findings at necropsy were unremarkable for all four dams.

No significant findings were recorded for clinical signs, detailed physical examination and arena observations. Underactive behaviour in males and females and unsteady posture in females were observed briefly during Week 1 in animals at 1000 mg/kg/day and dose related increases in post dosing salivation and chin rubbing were seen. Behavioural testing during Week 5 of dosing, including sensory reactivity findings, grip strength values and motor activity scores showed no differences considered to be associated with test material.

During late gestation females receiving 1000 mg/kg/day showed significantly lower weight gain than Controls but bodyweight and bodyweight gain were unaffected during lactation.Females receiving 1000 mg/kg/day showed lower food consumption during late gestation (Days 14-19) corresponding with the period of lower bodyweight gain. Food consumption during lactation was not significantly affected.

There was no effect on oestrous cycles, precoital interval, mating performance or fertility. All females mated and were pregnant but at 1000 mg/kg/day there was evidence of increased sensitivity in late gestation: two females had to be terminated before giving birth and one female was terminated during parturition, leading to a reduction in the gestation index. Gestation length was within normal range but there was a slight increase in the numbers of animals having longer (23 day) gestation periods. All animals in the Control, 100 and 300 mg/kg/day groups gave birth to a live litter. There was no effect of test material on the number of implantations but at 1000 mg/kg/day post implantation survival index, live birth index and viability index were all lower than Control so that live litter size was smaller. Lactation index assessed on Day 7 of lactation was unaffected. There was no effect at dose levels of 300 mg/kg/day or below. Male and female offspring bodyweights were not adversely affected.

Among the Toxicity subgroup animals, males receiving 1000 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. Bodyweight during the recovery phase was similar to controls. Liver weights were higher in males and females receiving 300 or 1000 mg/kg/day and kidney weights were higher than Control in females at 1000 mg/kg/day. Organ weight measurement two weeks after the end of the dosing period showed that the effects had been reversed and organs were normal size.  Biochemical changes in females at 1000 mg/kg/day, such as increased alanine aminophosphatase, alanine aminotransferase, cholesterol and bilirubin levels also suggest that the metabolic function of the liver may have been altered by administration of the test item. All the above discussed parameters showed complete recovery after 2 weeks.

Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for males was 300 mg/kg/day, the NOAEL for maternal toxicity was 300 mg/kg/day and the NOAEL for reproduction/developmental toxicity was at least 300 mg/kg/day. The NOAEL for unmated females was 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

981 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well conducted and well described studies in accordance with GLP and OECD Guidelines 422 (1996) and 408 without any deviation.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a 28 days combined repeated dose and reproduction screening study, males receiving 1000 mg/kg bw/day showed lower overall weight gain (Week 0-5) compared with control. Bodyweight during the recovery phase was similar to controls. The liver was identified as a target organ in this study, to a greater extent in females than males. Liver weight increases were recorded at 300 mg/kg bw/day and 1000 mg/kg bw/day. Biochemical changes in females at 1000 mg/kg bw/day, such as increased alanine aminophosphatase, alanine aminotransferase, cholesterol and bilirubin levels also suggest that the metabolic function of the liver may have been altered by administration of CAS 106185-75-5. All the above discussed parameters showed complete recovery after 2 weeks and are therefore considered to be adaptative changes. In pregnant females, continued administration of 1000 mg/kg bw/day resulted in loss of condition of some animals in late gestation and in the premature termination of three females. There was no effect on oestrous cycles, precoital interval, mating performance or fertility. All females were mated and pregnant.

Also, in a 13-week repeated dose toxicity study, males and/or females exposed by diet to the substance showed slight non-adverse and reversible changes in haematological parameters (erythrocyte and haemoglobin concentrations, prothrombin time and haematocrit), biochemical parameters (urea, blood urea nitrogen and creatinine concentrations, glucose,alkaline phosphatase, albumin concentrations and albumin/globulin ratio), associated with increases in liver and/or kidney weights. These observations are thought to be indicative of adaptations of metabolism/excretion in the liver and kidneys, with absence of degenerative or functional change in liver or kidneys. No effects on reproductive organs were observed either in males or females up to the highest dose tested, equivalent to 981 mg/kg bw/day for males and 1109 mg/kg bw/day for females.

Effects on developmental toxicity

Description of key information

A GLP study was conducted according to OECD Guideline 414. 




  
  

NOAEL for maternal toxicity = 300 mg/kg bw/day; 






NOAEL for embryo-fetal development = 750 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March - 13 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

24 April 2014

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): (2E)-ETHYL-4-(2,2,3-TRIMETHYLCYCLOPENT-3-EN-1-YL) BUT-2-EN-1-OL
- IUPAC name: 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-(2E)-buten-1-ol
- CAS No: 106185-75-5
- Common CAS No: 28219-61-6
- EC No: 248-908-8
- Physical state: colourless to very pale yellow liquid
- Analytical purity: trans-isomer 92.4 %
- Impurities: cis-isomer 5.6%, others 2.0%
- Purity test date: 30 October 2013
- Lot/batch No.: 147592
- Expiration date of the lot/batch: 28 October 2015
- Storage condition of test material: Refrigerated (2-8 °C), protected from light and under nitrogen

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: 231-282 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods; Grid bottomed cages were used during pairing.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: ca. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: Approximately 50 % of the final volume of vehicle was added to the required amount of test substance and magnetically stirred to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
- Frequency of preparation: Weekly
- Storage of preparation: Refrigerated (nominally 2-8 °C)

VEHICLE
- Concentration in vehicle: 20, 60 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: The homogeneity and stability of formulations in the concentration range 1 to 200 mg/mL were confirmed for 15 days following refrigerated storage (2-8 °C) and for two days following ambient storage (nominally 21 ˚C) as part of another study, Huntingdon Life Sciences (OAD0002).

Achieved concentration: Samples of each formulation prepared for administration on Day 1 and last day of dosing were analysed for achieved concentration of the test substance.
Results: The mean concentrations of test substance in test formulations analysed for the study were within 3 % of nominal concentrations, confirming accurate formulation.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: Ejected copulation plug / sperm in vaginal smear referred to as Day 0 of pregnancy.
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

14 days (Days 6-19 p.c.)

Frequency of treatment:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for investigation in this study (0, 100, 300 and 750 mg/kg bw/day) were selected in conjunction with the Sponsor, based on the results of a previously conducted combined repeated dose toxicity study with reproductive/developmental toxicity screening test in the rat (Huntingdon Life Sciences Report No. OAD0002). Considering the signs of excessive maternal toxicity at 1000 mg/kg bw/day, the high dose level for this embryo fetal development study was set at 750 mg/kg bw/day. The doses of 100 and 300 mg/kg bw/day were selected as the low and intermediate dose levels to establish a suitable dose relationship for any treatment-related changes and to give a 3 and 2.5-fold interval between doses, respectively.
- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s).
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.
Detailed observations were recorded daily at the following times in relation to dose administration:
At the end of dosing of each group; one to two hours after completion of dosing of all groups; as late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and daily from Days 6 to 20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION: Not recorded

POST-MORTEM EXAMINATIONS: Yes
- Animals were killed by carbon dioxide asphyxiation on Day 20 after mating.
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorption sites: Yes
- Number of late resorption sites: Yes
- Number of live fetuses: Yes
- Number of dead fetuses: Yes

Fetal examinations:

Fetuses were killed by chilling on a cool plate (approximately 0 °C).

Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning for visceral abnormalities. IMS fixed fetuses were processed and stained with Alizarin Red for skeletal development and abnormalities.

Statistics:

See "Any other information on materials and methods incl. tables"

Indices:

Reproductive assessment
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses)/ Number of implantations] x 100

Historical control data:

Historical background data was used to compare the incidences of developmental toxicity.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Signs in relation to dose administration were limited to the 750 mg/kg bw/day group where a low incidence of piloerection (from Day 8-19 of gestation) and underactive behaviour (from Day 13-17 of gestation) were observed 1-2 hours after dose administration but on some occasions the signs persisted to the end of the working day; two females showed hunched posture on a single occasion.
- A dose-dependent incidence of salivation or chin rubbing was also apparent among females receiving 300 or 750 mg/kg bw/day from Day 7/8 of gestation. These findings are commonly observed following oral (gavage) administration and are considered to reflect distaste of the test formulation rather than a direct effect of treatment.
- At routine physical examination, treatment-related signs were limited to a single female receiving 750 mg/kg bw/day which showed signs of piloerection and reduced body tone immediately prior to despatch to necropsy for scheduled termination on Day 20 of gestation. There were no other treatment-related signs observed at routine physical examination.

Mortality:

no mortality observed

Description (incidence):

- Oral administration of test substance to pregnant CD rats was well tolerated and there were no premature deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Females receiving 750 mg/kg bw/day showed statistically significant mean body weight loss of 3 g (compared to mean body weight gain of 3 g in the Control group) between Day 6 and Day 7 of gestation, and statistically significant lower mean body weight gain between Day 12 and Day 13 of gestation (3 g versus 8 g in the Control group). Slightly low weight gain was also apparent from Days 17-20 of gestation (36 g versus 46 g in the Control group). On all other recording occasions, mean body weight gain was essentially similar to or slightly higher than Controls, however overall mean weight gain from Day 6 to Day 20 of gestation was statistically significantly lower than Control (-13 %). There was no effect of treatment on the weight gain of females receiving 100 or 300 mg/kg bw/day.
- At scheduled termination on Day 20 of gestation, the mean gravid uterine weight of females in the 750 mg/kg bw/day group was slightly lower than Control (-7 %) and when overall mean body weight gain was adjusted for the contribution of the gravid uterus, net body weight gain during gestation was statistically significantly lower than Control (-36 %). Mean gravid uterine weight and net mean body weight gain were similar to Control for females receiving 100 or 300 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Following the commencement of treatment on Day 6 of gestation, mean food intake among females in the 750 mg/kg bw/day group was slightly but statistically significantly lower (overall decrease of -12 %) than Control throughout the treatment period. There was no effect of treatment on the mean food intake of females receiving 100 or 300 mg/kg bw/day.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of post-implantation losses was statistically significantly lower in all treated groups when compared to Controls. All values were within the Historical Control Data (HCD) range and in view of the direction of change, these minor differences were considered fortuitous and unrelated to treatment.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- All females were found to be pregnant with live young at scheduled termination on Day 20 of gestation.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of early resorptions was statistically significantly lower in all treated groups when compared to Controls. All values were within the Historical Control Data (HCD) range and in view of the direction of change, these minor differences were considered fortuitous and unrelated to treatment.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality:
- Oral administration of test substance to pregnant CD rats was well tolerated and there were no premature deaths.

Clinical signs:
- Signs in relation to dose administration were limited to the 750 mg/kg bw/day group where a low incidence of piloerection (from Day 8-19 of gestation) and underactive behaviour (from Day 13-17 of gestation) were observed 1-2 hours after dose administration but on some occasions the signs persisted to the end of the working day; two females showed hunched posture on a single occasion.
- A dose-dependent incidence of salivation or chin rubbing was also apparent among females receiving 300 or 750 mg/kg bw/day from Day 7/8 of gestation. These findings are commonly observed following oral (gavage) administration and are considered to reflect distaste of the test formulation rather than a direct effect of treatment.
- At routine physical examination, treatment-related signs were limited to a single female receiving 750 mg/kg bw/day which showed signs of piloerection and reduced body tone immediately prior to despatch to necropsy for scheduled termination on Day 20 of gestation. There were no other treatment-related signs observed at routine physical examination.

Body weight:
- Females receiving 750 mg/kg bw/day showed statistically significant mean body weight loss of 3 g (compared to mean body weight gain of 3 g in the Control group) between Day 6 and Day 7 of gestation, and statistically significant lower mean body weight gain between Day 12 and Day 13 of gestation (3 g versus 8 g in the Control group). Slightly low weight gain was also apparent from Days 17-20 of gestation (36 g versus 46 g in the Control group). On all other recording occasions, mean body weight gain was essentially similar to or slightly higher than Controls, however overall mean weight gain from Day 6 to Day 20 of gestation was statistically significantly lower than Control (-13 %). There was no effect of treatment on the weight gain of females receiving 100 or 300 mg/kg bw/day.
- At scheduled termination on Day 20 of gestation, the mean gravid uterine weight of females in the 750 mg/kg bw/day group was slightly lower than Control (-7 %) and when overall mean body weight gain was adjusted for the contribution of the gravid uterus, net body weight gain during gestation was statistically significantly lower than Control (-36 %). Mean gravid uterine weight and net mean body weight gain were similar to Control for females receiving 100 or 300 mg/kg bw/day.

Food consumption:
- Following the commencement of treatment on Day 6 of gestation, mean food intake among females in the 750 mg/kg bw/day group was slightly but statistically significantly lower (overall decrease of -12 %) than Control throughout the treatment period. There was no effect of treatment on the mean food intake of females receiving 100 or 300 mg/kg bw/day.

Gross pathology:
- There were no treatment-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

Litter responses:
- All females were found to be pregnant with live young at scheduled termination on Day 20 of gestation.

Reproductive assessment:
- There was no effect of maternal treatment with test substance at any dose level investigated on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- or post-implantation losses. Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations.
- More particularly, early resorptions and post-implantation loss were statistically significantly reduced in all treated groups compared to control group but they cannot be considered as an effect of the treatment as they are within the Historical Control Data (HCD).

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

effects observed, treatment-related

Localisation:

other: Food intake, body weight, and uterus weight

Description (incidence and severity):

The mean food intake of females receiving 750 mg/kg/day was slightly but statistically
significantly lower than Control. The mean weight gain during gestation was
13% lower than Control, mean gravid uterine weight was 7% lower than Control and net
weight gain when adjusted for the contribution of the gravid uterus was 36% lower than
Control.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

In all treated groups, a dose dependent marginal decrease in mean male, female and overall fetal weights was apparent with statistical significance and values slightly below the lower limit of the HCD attained in the 300 and 750 mg/kg bw/day groups. As there is no other effects observed, this light decrease could be considered as a non adverse effect.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): In all treated groups, a dose dependent marginal decrease in mean male, female and overall fetal weights was apparent with values slightly below the lower limit of the HCD range in all treated groups and statistical significance attained in the 300 and 750 mg/kg/day groups. As there is no other effects observed, this light decrease could be considered as a non adverse effect.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

Description (incidence and severity):

not relevant for OECD 414

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Across all treated groups there was a slight increase in the incidence of incomplete ossification of sternebrae, sacrocaudal vertebral arches and pelvic bones compared to concurrent control; the mean percentage of fetuses per litter with affected
sternebrae in the 100 and 750 mg/kg/day groups (73.7% and 85.2%, respectively) exceeded the upper limit of the Historical Control Data (HCD) of 69.2%. In addition, the fetal incidence of incomplete ossification of the pelvic bones in the 750 mg/kg/day exceeded the upper limit of the HCD range (12 fetuses in 10 litters). This incomplete ossification was considered likely to relate to the marginal decrease in mean fetal weight throughout the treated groups, representing a slight delay in maturation.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Placental, litter and fetal weights:
- In all treated groups, a dose dependent marginal decrease in mean male, female and overall fetal weights was apparent with statistical significance and values slightly below the lower limit of the HCD attained in the 300 and 750 mg/kg bw/day groups.
- Mean placental weights were unaffected by maternal treatment in all treated groups.

Fetal pathology:
- There was no effect of treatment on the incidence of major abnormalities or minor visceral abnormalities. Across all treated groups there was a slight increase in the incidence of incomplete ossification of sternebrae, sacrocaudal vertebral arches and pelvic bones compared to concurrent control; the mean percentage of fetuses per litter with affected sternebrae in the 100 and 750 mg/kg bw/day groups (73.7 % and 85.2 %, respectively) exceeded the upper limit of the Historical Control Data (HCD) of 69.2 %. In addition, the fetal incidence of incomplete ossification of the pelvic bones in the 750 mg/kg bw/day exceeded the upper limit of the HCD range (12 fetuses in 10 litters). This incomplete ossification was considered likely to relate to the marginal decrease in mean fetal weight, especially at 750 mg/kg bw/day, representing a slight delay in maturation.
A slight increase in the incidence of 13/14 or 14/14 lumbar ribs was also observed across all treated groups compared to concurrent control; the fetal incidence in the 300 and 750 mg/kg bw/day groups slightly exceeded the upper limit of the HCD range (18 fetuses in 10 litters).

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect of the treatment

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 7.8.2/1: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) during gestation

 

Dose (mg/kg bw/day)		Body weight on Day 6	Terminal body weight on Day 20	Body weight change Days 6-20	Gravid uterine weight	Adjusted body weight Day 20	Adjusted body weight Day 20
Statistical test		Av	Wi	Wi	Wi	Wi	Wi
0 (control)	Mean	288	412	124	91	321	33
	SD	13.6	23.8	15.1	15.7	14.7	8.6
	N	20	20	20	20	20	20
100	Mean	291	416	125	94	322	31
	SD	13.8	23.4	14.0	11.2	18.7	9.6
	N	20	20	20	20	20	20
300	Mean	289	414	124	94	320	30
	SD	15.3	24.4	13.0	12.0	17.3	6.7
	N	20	20	20	20	20	20
750	Mean	285	391**	106**	85	306*	21**
	SD	16.6	23.8	15.4	8.8	20.5	12.4
	N	20	20	20	20	20	20

 

Table 7.8.2/2: Food consumption - group mean values (g/animal/day) during gestation

 

Dose (mg/kg bw/day)		Day 0-2	Day 3-5	Day 6-9	Day 10-13	Day 14-17	Day 18-19
Statistical test		Av	Av	Wi	Wi	Wi	Wi
0 (control)	Mean	25	26	22	25	28	27
	SD	2.6	2.9	2.2	2.5	2.4	2.5
	N	20	20	20	20	20	20
100	Mean	25	26	23	25	28	27
	SD	2.7	2.4	2.4	2.6	2.6	3.6
	N	20	20	20	20	20	20
300	Mean	25	27	22	25	27	27
	SD	1.9	2.3	2.7	2.5	2.1	2.6
	N	20	20	20	20	20	20
750	Mean	24	25	19**	23*	25**	23**
	SD	2.8	2.9	2.6	3.3	3.3	4.6
	N	20	20	20	20	20	20

 

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi: Treated groups compared with Control using Williams’ test.

* p<0.05; ** p<0.01

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 300 mg/kg bw/day. The NOAEL for embryo-fetal development was considered to be 750 mg/kg bw/day in rats.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, (2E)-ETHYL-4-(2,2,3-TRIMETHYLCYCLOPENT-3-EN-1-YL) BUT-2-EN-1-OL was administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (20/dose) at the dose levels of 0, 100, 300 and 750 mg/kg bw/day from Days 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

Oral administration of test substance to pregnant CD rats from Day 6 to Day 19 of gestation at doses up to 750 mg/kg bw/day was well tolerated and there were no mortalities. Signs in relation to dose administration were limited to the 750 mg/kg bw/day group where a low incidence of piloerection and underactive behaviour were observed 1-2 hours after dose administration but on some occasions the signs persisted to the end of the working day; two other females showed hunched posture on a single occasion. At routine physical examination, treatment-related signs were limited to a single female receiving 750 mg/kg bw/day which showed signs of piloerection and reduced body tone immediately prior to despatch to necropsy for scheduled termination on Day 20 of gestation. The mean food intake of females receiving 750 mg/kg bw/day was slightly but statistically significantly lower than Control throughout the treatment period and was associated with lower mean body weight during the same period, occasional periods of slightly reduced mean body weight gain, and mean body weight loss following the administration of the first dose. Overall mean weight gain during gestation was statistically significantly lower (-13 %) than Control, mean gravid uterine weight was 7 % lower than Control and net weight gain when adjusted for the contribution of the gravid uterus was 36 % lower than Control. There was no effect of treatment with test substance on the body weight gain, gravid uterine weight or food intake or liver weight of females receiving 100 or 300 mg/kg bw/day. There were no treatment-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

 

Litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- or post-implantation losses and sex ratio were unaffected by maternal treatment. In all treated groups mean fetal weights were marginally low when compared with Controls, with statistical significance attained in the 300 and 750 mg/kg bw/day groups. Mean placental weights were unaffected by maternal treatment at all dose levels investigated.

 

There was considered to be no adverse effect of maternal treatment on embryo-fetal development, and the incidence of major and minor visceral abnormalities showed no relationship to maternal treatment. In all treated groups there was a slightly higher incidence of incompletely ossified sternebrae, sacrocaudal vertebral arches and pelvic bones compared to concurrent control, which was considered likely to relate to marginally low mean fetal weight. The mean percentage of fetuses per litter with affected sternebrae in the 100 and 750 mg/kg bw/day groups (73.7 % and 85.2 %, respectively) exceeded the upper limit of the Historical Control Data (HCD) of 69.2 %. In addition, the fetal incidence of incomplete ossification of the pelvic bones in the 750 mg/kg bw/day exceeded the upper limit of the HCD range (12 fetuses in 10 litters). These minor skeletal variants were considered to reflect a slight delay in development, indicated by the marginally decreased mean fetal body weight in the treated groups. A slight increase in the incidence of 13/14 or 14/14 lumbar ribs was also observed across all treated groups compared to concurrent control; the fetal incidence in the 300 and 750 mg/kg bw/day groups slightly exceeded the upper limit of the HCD range (18 fetuses in 10 litters).

 

Therefore, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 300 mg/kg bw/day. The NOAEL for embryo-fetal development was considered to be 750 mg/kg bw/day in rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Well conducted and well described study in accordance with GLP and OECD Guideline 414 without any deviation.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, the test item was administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (20/dose) at the dose levels of 0, 100, 300 and 750 mg/kg bw/day from Days 6 to 19 after mating.

The mean food intake of females receiving 750 mg/kg bw/day was slightly but statistically significantly lower than Control throughout the treatment period and was associated with statistically significantly lower overall mean weight gain during gestation (-13 %), lower mean gravid uterine weight (-7 %) and lower net weight gain when adjusted for the contribution of the gravid uterus (-36 %).There was no effect of treatment with test substance on the body weight gain, gravid uterine weight or food intake or liver weight of females receiving 100 or 300 mg/kg bw/day. There were no treatment-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

Litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- or post-implantation losses and sex ratio were unaffected by maternal treatment. In all treated groups mean fetal weights were marginally low when compared with Controls, with statistical significance attained in the 300 and 750 mg/kg bw/day groups. Mean placental weights were unaffected by maternal treatment at all dose levels investigated.

There was considered to be no adverse effect of maternal treatment on embryo-fetal development, and the incidence of major and minor visceral abnormalities showed no relationship to maternal treatment.

Therefore, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 300 mg/kg bw/day.

The NOAEL for embryo-fetal development was considered to be 750 mg/kg bw/day in rats.

Justification for selection of Effect on developmental toxicity: via oral route:
This is the only study available for this endpoint.

Justification for classification or non-classification

In recent GLP repeated dose toxicity studies (OECD guideline 422 and 408 studies), only slight adaptative and reversible systemic toxicity changes were observed in males and females rats exposed to the registered substance up to the highest dose tested.

Furthermore, no effects on fertility, on or via lactation or on reproduction parameters were reported in the combined repeated dose and reproductive/developmental toxicity screening test and no effects on reproductive organs were detected in the 90-day repeated dose toxicity study.

The only adverse effects were observed in gestating females at 750 mg/kg bw/day in a OECD guideline 414 study and at 1000 mg/kg bw/day in an OECD guideline 422 study, therefore the NOAEL for maternal toxicity was set at 300 mg/kg bw/day.

Therefore, the registered substance does not need to be classified according to CLP Regulation (EC) n° 1272/2008.