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Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 094678
Purity : 92.8%
Name of test material (as cited in study report): 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol
Physical state: liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Date of manufacture: 25 November 2009
Expiry Date: 24 November 2011
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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
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:
no effects observed
Description (incidence and severity):
There were no significant necropsy findings for females on Day 7 of lactation.
Neuropathological findings:
not examined
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 / performance (P0)

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).

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.