Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-09-2009 to 06-05-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-09-2009 to 06-05-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: Stable.
- Solubility and stability of the test substance in the solvent/vehicle: Formulations up to the maximum dose concentration 150 mg/kg in Corn Oil was achieved during formulation analysis. Formulations were found to be stable in Corn Oil, when assessed following storage at ambient temperature (nominally 21°C) for up to seven days.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required amount of test item was weighed into a glass beaker on a tared precision balance and approximately 80%
of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item within Corn Oil BP.
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.
Species:
rat
Strain:
other: Han Wistar: RccHan: WIST
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 421 and the other relevant guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: (P) Males ca. 11 wks ; Females ca. 11 wks
- Weight at study initiation: (P) Males: 307 - 349 g; Females: 192 - 215 g
- Fasting period before study: No.
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: > 7 days before commencement of treatment. Females: > 7 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Feed: Certified pelleted diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: Males: From: 2009-10-01 To: 2009-11-26 / Females: From: 2009-10-01 To: 2009-11-26
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Corn Oil. The stability and homogeneity of the test item formulations were determined. Formulations up to the maximum dose concentration 150 mg/kg in Corn Oil was achieved during formulation analysis. Formulations were found to be stable in Corn Oil, when assessed following storage at ambient temperature (nominally 21°C) for up to seven days.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Corn Oil was considered as appropriate based on test item solubility.
- Concentration in vehicle: application formulations investigated during the study were found to comprise test item in the range of 88.0% to 97.2% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 1.7% (< 15%) from the corresponding mean. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Purity: Corn Oil (certified – batch number indicated in the full study report)
- Other: Dose-formulations were analysed during the study and were reported as with ± 20% applied limits.
Details on mating procedure:
- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plug and sperm in vaginal smear. Day 0 of gestation was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed. Formulations up to the maximum dose concentration 150 mg/kg in Corn Oil was achieved during formulation analysis. Formulations were found to be stable in Corn Oil, when assessed following storage at ambient temperature (nominally 21°C) for up to seven days.
- The analysis consisted of GC FID analysis with external calibration within a dedicated formulation analysis report attached to the full study report. Samples of Corn Oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations along with internal standard for the calibration standards (for samples: along with specific dilution factors to place samples into the calibration range). These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). linearity = 0.9985 and repeatability (n=8) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 89.5% (n=3) at 2 mg/mL and 96.5% (n=3) at 30 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 20% applied limits for nominal concentrations and 10% for time-zero (homogeneity mean) confirming accurate test item/vehicle formulation.
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until day 3 post-partum.
Frequency of treatment:
Daily; at approximately the same time each day.
F1 generation were not dosed.
Details on study schedule:
- Age at mating of the mated animals in the study: F0 generation: Males: ca. 13 weeks ; Females: ca. 13 weeks. (i.e. after two weeks treatment).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Vehicle Control Group
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2 - Low Treatment Group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 3 - Intermediate Treatment Group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 4 - High Treatment Group
No. of animals per sex per dose:
10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 28-day toxicity study (Report number attached to and cited in the full study report).
- Rationale for animal assignment (if not random): Randomly assigned
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (and during acclimatisation period, once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of
difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily until necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was monitored during the study: Males: Weekly during pre-pairing and after pairing periods ; Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 – 21 post coitum, and days 1 - 4 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable.
- Time schedule for examinations: Not applicable.
Oestrous cyclicity (parental animals):
Daily smears
Sperm parameters (parental animals):
Parameters examined in F0 male parental generations:
testis weight, epididymis weight and histology/histopathology

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: clinical observations, litter size, sex ratio, survival indices, body weight change, macroscopic pathology / abnormalities. Histopathology.

GROSS EXAMINATION OF DEAD PUPS:
Yes, where required or if applicable.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: F0 Males: After 4 weeks treatment.
- Maternal animals: F0 females failing to produce a viable litter: On day 4 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively. For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed at scheduled intervals.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at ca. 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Particular attention was paid to the external genitalia.

HISTOPATHOLOGY / ORGAN WEIGHTS
The thyroid tissues were prepared for microscopic examination from one male and one female offspring in each litter. Organ weights were not performed for F1 males or F1 females.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
Means and standard deviations of various data were calculated.
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Mating performance, fertility, gestation length, gestation index
Offspring viability indices:
Post-implantation survival index, viability index, lactation index and sex ratio
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day: clinical findings included pushing the head through the bedding after dosing. In addition, salivation before and/or after dosing was observed in isolated individuals.

At 30 mg/kg bw/day: two dams occasionally had salivation after dosing. No clinical signs or observations were observed in the males during the study at this dose level.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths in the F0 generation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and body weight gain were not affected by treatment with the test item in males or females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day: females, mean food consumption was reduced during the lactation period without statistical significance for the overall group (-22.6% in comparison to the control group). However, marked reduced food consumption during the lactation period was observed with 3 individual females (#73, 76, and 80) in this group.
Food efficiency:
not examined
Description (incidence and severity):
See 'Food consumption'
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
At 150 mg/kg bw/day: multifocal progressive cardiomyopathy was observed in the heart of all females. This was considered to be adverse in nature. Centrilobular hypertrophy was noted in the liver in 5/10 males and 5/10 females. In addition, hematopoiesis was increased in the females. Both these findings in the liver were considered to be adaptive and not adverse in nature. In some females, a periportal fatty change was observed. There was an increased incidence of thymic involution in the females, which was considered to be related to stress and not directly to the test item.

At 30 mg/kg bw/day: There was an increased incidence of thymic involution in the females, which was considered to be related to stress and not directly to the test item. There was no test item related observations in males/females at this dose level.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no treatment related findings. No cell or stage specific abnormalities were noted.

No test item-related changes were observed in the reproductive organs or in the comparison of spermatogenesis and interstitial testicular structures.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was not affect on fertility or mating performance related to treatment.

Post-implantation loss was unaffected by treatment with the test item. The statistically significant increase in the total number of losses in group 2 (24 compared to 11 in the control group) was mainly due to litter #53, which had 10 post-implantation losses. Since there was no dosedependent pattern, this was an incidental occurrence and not related to treatment with the test item.

At 150 mg/kg bw/day : significant postnatal losses were seen after 4 days, notably in three litters (#73, 76, and 80), leading to an overall statistically significant increase in losses for this group.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs related to treatment.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day : significant postnatal losses were seen after 4 days, notably in three litters (#73, 76, and 80), leading to an overall statistically significant increase in losses for this group.
Applicant assessment indicates that this may be due to general toxicity effects in the P0 females at this dose level (poor maternal care).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day: body weight gain over days 1 - 4 and mean body weight on day 4 post partum were reduced compared to the control group.
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
Description (incidence and severity):
No findings were attributable to treatment.
Histopathological findings:
no effects observed
Description (incidence and severity):
No findings were attributable to treatment.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-effect level (NOEL) and the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 30 mg/kg body weight per day.
Executive summary:

The study was performed according the requirements of OECD TG 421 guideline under GLP conditions. The purpose of this study was to assess general systemic toxic potential in rats, including a screening test for reproductive / development effects, with administration of the test item by oral gavage administration for a least four weeks in Wistar Han : RccHan : WIST rats. Four groups, each comprising ten male and ten female rats, received test item at doses of 0 (control), 10, 30 or 150 mg/kg/day. The control group of ten males and ten females was similarly dosed with vehicle alone (Corn Oil), at the same dose volume as the treated groups. A standard dose of 5 mL/kg bw with daily adjustment for actual bodyweight was used. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum. The F1 generation received no direct administration of the test item; any exposure, if it occurred was in utero or via the milk. The mean concentrations of the test item in test formulations were analyzed for the study and were within ±20% of nominal concentrations, confirming accurate formulation. The test item was determined to be stable at ambient storage for 7 days corn oil formulations. At 150 mg/kg bw/day: clinical findings included pushing the head through the bedding after dosing. In addition, salivation before and/or after dosing was observed in isolated individuals and females, mean food consumption was reduced during the lactation period without statistical significance for the overall group (-22.6% in comparison to the control group). However, marked reduced food consumption during the lactation period was observed with 3 individual females (#73, 76, and 80) in this group. At 30 mg/kg bw/day: two dams occasionally had salivation after dosing. No clinical signs or observations were observed in the males during the study at this dose level. At 150 mg/kg bw/day and 30 mg/kg bw/day. Reduced size of thymus and accentuated lobular pattern in liver were observed as gross lesions in some females. Histopathological examinations indicated microscopic thymic and/or liver changes. However, during histopathology there was an increased incidence of thymic involution in the females, which was considered to be related to stress and not directly to the test item. Significant other histopathology findings were confined to 150 mg/kg bw/day where multifocal progressive cardiomyopathy was observed in the heart of all females. This was considered to be adverse in nature. Centrilobular hypertrophy was noted in the liver in 5/10 males and 5/10 females. In addition, hematopoiesis was increased in the females. Both these findings in the liver were considered to be adaptive and not adverse in nature. In some females, a periportal fatty change was observed. No test item-related changes were observed in the reproductive organs or in the comparison of spermatogenesis and interstitial testicular structures. In the F1 generation, at 150 mg/kg bw/day : significant postnatal losses were seen after 4 days, notably in three litters (#73, 76, and 80), leading to an overall statistically significant increase in losses for this group. Additionally, body weight gain over days 1 - 4 and mean body weight on day 4 post partum were reduced compared to the control group. Applicant assessment indicates that this may be due to general toxicity effects in the P0 females at this dose level (poor maternal care). During external examinations and examination of sex ratios indicated no effects in relation to treatment. Under the conditions of this study, the no-observed-effect level (NOEL and the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 30 mg/kg body weight per 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:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
460-490-0
EC Name:
-
Cas Number:
477218-42-1
Molecular formula:
C18H32O3
IUPAC Name:
2-[(1S)-1-[(1R)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate; 2-[(1S)-1-[(1S)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate
Test material form:
liquid
Details on test material:
Physical state: Liquid
- Storage condition of test material: Room temperature (15 to 25°C), protected from light
- Other: colourless liquid
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: Stable.
- Solubility and stability of the test substance in the solvent/vehicle: Formulations up to the maximum dose concentration 150 mg/kg in Corn Oil was achieved during formulation analysis. Formulations were found to be stable in Corn Oil, when assessed following storage at ambient temperature (nominally 21°C) for up to seven days.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required amount of test item was weighed into a glass beaker on a tared precision balance and approximately 80%
of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item within Corn Oil BP.
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.

Test animals

Species:
rat
Strain:
other: Han Wistar: RccHan: WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: (P) Males ca. 11 wks ; Females ca. 11 wks
- Weight at study initiation: (P) Males: 307 - 349 g; Females: 192 - 215 g
- Fasting period before study: No.
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: > 7 days before commencement of treatment. Females: > 7 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Feed: Certified pelleted diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: Males: From: 2009-10-01 To: 2009-11-26 / Females: From: 2009-10-01 To: 2009-11-26

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Corn Oil. The stability and homogeneity of the test item formulations were determined. Formulations up to the maximum dose concentration 150 mg/kg in Corn Oil was achieved during formulation analysis. Formulations were found to be stable in Corn Oil, when assessed following storage at ambient temperature (nominally 21°C) for up to seven days.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Corn Oil was considered as appropriate based on test item solubility.
- Concentration in vehicle: application formulations investigated during the study were found to comprise test item in the range of 88.0% to 97.2% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 1.7% (< 15%) from the corresponding mean. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Purity: Corn Oil (certified – batch number indicated in the full study report)
- Other: Dose-formulations were analysed during the study and were reported as with ± 20% applied limits.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed. Formulations up to the maximum dose concentration 150 mg/kg in Corn Oil was achieved during formulation analysis. Formulations were found to be stable in Corn Oil, when assessed following storage at ambient temperature (nominally 21°C) for up to seven days.
- The analysis consisted of GC FID analysis with external calibration within a dedicated formulation analysis report attached to the full study report. Samples of Corn Oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations along with internal standard for the calibration standards (for samples: along with specific dilution factors to place samples into the calibration range). These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). linearity = 0.9985 and repeatability (n=8) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 89.5% (n=3) at 2 mg/mL and 96.5% (n=3) at 30 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 20% applied limits for nominal concentrations and 10% for time-zero (homogeneity mean) confirming accurate test item/vehicle formulation.
Details on mating procedure:
- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plug and sperm in vaginal smear. Day 0 of gestation was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding.
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until day 3 post-partum.
Frequency of treatment:
Daily; at approximately the same time each day.
F1 generation were not dosed.
Duration of test:
- Age at mating of the mated animals in the study: F0 generation: Males: ca. 13 weeks ; Females: ca. 13 weeks. (i.e. after two weeks treatment).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Vehicle Control Group
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2 - Low Treatment Group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 3 - Intermediate Treatment Group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 4 - High Treatment Group
No. of animals per sex per dose:
10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 28-day toxicity study (Report number attached to and cited in the full study report).
- Rationale for animal assignment (if not random): Randomly assigned

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (and during acclimatisation period, once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of
difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily until necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was monitored during the study: Males: Weekly during pre-pairing and after pairing periods ; Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 – 21 post coitum, and days 1 - 4 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable.
- Time schedule for examinations: Not applicable.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
Means and standard deviations of various data were calculated.
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Indices:
Offspring viability indices including; post-implantation survival index, live birth index, viability index, lactation index and sex ratio.
Historical control data:
Historical control data was available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day: clinical findings included pushing the head through the bedding after dosing. In addition, salivation before and/or after dosing was observed in isolated individuals.

At 30 mg/kg bw/day: two dams occasionally had salivation after dosing. No clinical signs or observations were observed in the males during the study at this dose level.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths in the F0 generation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and body weight gain were not affected by treatment with the test item in males or females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day: females, mean food consumption was reduced during the lactation period without statistical significance for the overall group (-22.6% in comparison to the control group). However, marked reduced food consumption during the lactation period was observed with 3 individual females (#73, 76, and 80) in this group.
Food efficiency:
not examined
Description (incidence and severity):
See 'Food consumption'
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute weights and the organ/body weight ratios of the testes and epididymides were similar in all dose groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day: Reduced size of thymus and accentuated lobular pattern in liver were observed as gross lesions in 4/10 females. Histopathological examinations indicated microscopic thymic and/or liver changes. See below.

At 30 mg/kg bw/day: Reduced size of thymus and accentuated lobular pattern in liver were observed as gross lesions in 3/10 females. Histopathological examinations indicated microscopic thymic and/or liver changes. See below.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day: multifocal progressive cardiomyopathy was observed in the heart of all females. This was considered to be adverse in nature. Centrilobular hypertrophy was noted in the liver in 5/10 males and 5/10 females. In addition, hematopoiesis was increased in the females. Both these findings in the liver were considered to be adaptive and not adverse in nature. In some females, a periportal fatty change was observed. There was an increased incidence of thymic involution in the females, which was considered to be related to stress and not directly to the test item.

At 30 mg/kg bw/day: There was an increased incidence of thymic involution in the females, which was considered to be related to stress and not directly to the test item. There was no test item related observations in males/females at this dose level.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation loss was unaffected by treatment with the test item. The statistically significant increase in the total number of losses in group 2 (24 compared to 11 in the control group) was mainly due to litter #53, which had 10 post-implantation losses. Since there was no dosedependent pattern, this was an incidental occurrence and not related to treatment with the test item.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
At up to 150 mg/kg bw/day: gestation length and gestation index were unaffected by treatment.
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic

Maternal abnormalities

Abnormalities:
no effects observed
Description (incidence and severity):
No treatment related Adverse Effects observed up to the maximum dose level

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
At 150 mg/kg bw/day : significant postnatal losses were seen after 4 days, notably in three litters (#73, 76, and 80), leading to an overall statistically significant increase in losses for this group. Additionally, body weight gain over days 1 - 4 and mean body weight on day 4 post partum were reduced compared to the control group. Applicant assessment indicates that this may be due to general toxicity effects in the P0 females at this dose level (poor maternal care).
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day : significant postnatal losses were seen after 4 days, notably in three litters (#73, 76, and 80), leading to an overall statistically significant increase in losses for this group. Additionally, body weight gain over days 1 - 4 and mean body weight on day 4 post partum were reduced compared to the control group. Applicant assessment indicates that this may be due to general toxicity effects in the P0 females at this dose level (poor maternal care).
External malformations:
no effects observed
Description (incidence and severity):
Examination of offspring did not reveal any findings attributable to treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring did not reveal any findings attributable to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring did not reveal any findings attributable to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in postnatal survival

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-effect level (NOEL) and the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 30 mg/kg body weight per day.
Executive summary:

The study was performed according the requirements of OECD TG 421 guideline under GLP conditions. The purpose of this study was to assess general systemic toxic potential in rats, including a screening test for reproductive / development effects, with administration of the test item by oral gavage administration for a least four weeks in Wistar Han : RccHan : WIST rats. Four groups, each comprising ten male and ten female rats, received test item at doses of 0 (control), 10, 30 or 150 mg/kg/day. The control group of ten males and ten females was similarly dosed with vehicle alone (Corn Oil), at the same dose volume as the treated groups. A standard dose of 5 mL/kg bw with daily adjustment for actual bodyweight was used. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum. The F1 generation received no direct administration of the test item; any exposure, if it occurred was in utero or via the milk. The mean concentrations of the test item in test formulations were analyzed for the study and were within ±20% of nominal concentrations, confirming accurate formulation. The test item was determined to be stable at ambient storage for 7 days corn oil formulations. At 150 mg/kg bw/day: clinical findings included pushing the head through the bedding after dosing. In addition, salivation before and/or after dosing was observed in isolated individuals and females, mean food consumption was reduced during the lactation period without statistical significance for the overall group (-22.6% in comparison to the control group). However, marked reduced food consumption during the lactation period was observed with 3 individual females (#73, 76, and 80) in this group. At 30 mg/kg bw/day: two dams occasionally had salivation after dosing. No clinical signs or observations were observed in the males during the study at this dose level. At 150 mg/kg bw/day and 30 mg/kg bw/day. Reduced size of thymus and accentuated lobular pattern in liver were observed as gross lesions in some females. Histopathological examinations indicated microscopic thymic and/or liver changes. However, during histopathology there was an increased incidence of thymic involution in the females, which was considered to be related to stress and not directly to the test item. Significant other histopathology findings were confined to 150 mg/kg bw/day where multifocal progressive cardiomyopathy was observed in the heart of all females. This was considered to be adverse in nature. Centrilobular hypertrophy was noted in the liver in 5/10 males and 5/10 females. In addition, hematopoiesis was increased in the females. Both these findings in the liver were considered to be adaptive and not adverse in nature. In some females, a periportal fatty change was observed. No test item-related changes were observed in the reproductive organs or in the comparison of spermatogenesis and interstitial testicular structures. In the F1 generation, at 150 mg/kg bw/day : significant postnatal losses were seen after 4 days, notably in three litters (#73, 76, and 80), leading to an overall statistically significant increase in losses for this group. Additionally, body weight gain over days 1 - 4 and mean body weight on day 4 post partum were reduced compared to the control group. Applicant assessment indicates that this may be due to general toxicity effects in the P0 females at this dose level (poor maternal care). During external examinations and examination of sex ratios indicated no effects in relation to treatment. Under the conditions of this study, the no-observed-effect level (NOEL and the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 30 mg/kg body weight per day.