2-Buten-1-ol, 2-methyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-, (2E)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2010 to 17 September 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

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

Justification for type of information:

See attached justification in section 13.2

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

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

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 oral 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

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

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
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

Observations and examinations performed and frequency:

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).

Sacrifice and pathology:

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.

Other examinations:

ESTROUS CYCLICITY (PARENTAL ANIMALS):
For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all Reproductive subgroup 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.

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.

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.

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):

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.

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):

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 examined

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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 CAS 28219-61-6.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Main phase males and unmated females: Bodyweight adjusted liver weights were higher than Control in males and females after five weeks dosing at 300 or 1000 mg/kg/day and kidney weights were higher than Control in females at 1000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarge livers were observed in females receiving 1000 mg/kg/day. Pale adrenals were observed in females receiving 300 and 1000 mg/kg/day with dose reponse apparent.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centilobular hepatocyte hypertrophy was seen in the liver the 2 females receiving 1000 mg/kg/day with enlarge livers. No histopathological findings were seen in the kidneys. No findings were seen in the adrenal glands.

Histopathological findings: neoplastic:

no effects observed

Details on results:

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.

GROSS PATHOLOGY (OFFSPRING): The type and distribution of findings at necropsy of the offspring that survived to scheduled termination on Day 7 of age did not suggest any adverse effect of treatment.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

None

Conclusions:

The No-Observed-Adverse–Effect-Level (NOAEL) for males and unmated females was 300 mg/k/g/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.

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 adverselyaffected.

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 and unmated females was 300 mg/k/g/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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-buten-1-ol

Cas Number:

106185-75-5

Molecular formula:

C14H24O

IUPAC Name:

(2E)-2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-buten-1-ol

Test animals

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
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

Examinations

Observations and examinations performed and frequency:

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).

Sacrifice and pathology:

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.

Other examinations:

ESTROUS CYCLICITY (PARENTAL ANIMALS):
For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all Reproductive subgroup 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.

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.

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.

Results and discussion

Results of examinations

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):

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.

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):

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 examined

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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 CAS 28219-61-6.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Main phase males and unmated females: Bodyweight adjusted liver weights were higher than Control in males and females after five weeks dosing at 300 or 1000 mg/kg/day and kidney weights were higher than Control in females at 1000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarge livers were observed in females receiving 1000 mg/kg/day. Pale adrenals were observed in females receiving 300 and 1000 mg/kg/day with dose reponse apparent.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Centilobular hepatocyte hypertrophy was seen in the liver the 2 females receiving 1000 mg/kg/day with enlarge livers. No histopathological findings were seen in the kidneys. No findings were seen in the adrenal glands.

Histopathological findings: neoplastic:

no effects observed

Details on results:

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.

GROSS PATHOLOGY (OFFSPRING): The type and distribution of findings at necropsy of the offspring that survived to scheduled termination on Day 7 of age did not suggest any adverse effect of treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The No-Observed-Adverse–Effect-Level (NOAEL) for males and unmated females was 300 mg/k/g/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.

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 adverselyaffected.

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 and unmated females was 300 mg/k/g/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.