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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016=2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study under GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study under GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
CiToxLab Hungary Ltd. H-8200 Veszprém, Szabadságpuszta, Hungary
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation:
- Fasting period before study: no, fasting only before sacrifice
- Housing: males housed in groups of 4, in polycarbonate cages. Females housed singly during and after mating. Lignocell (R) and Arbocel (R) natural crinklets
- Diet (e.g. ad libitum): at lib, ssniff (R)
- Water (e.g. ad libitum): ad lib, municipal water supply
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate vehicle for oily liquid test material
- Concentration in vehicle: 100, 300 and 1000 mg/kg bw/d
- Amount of vehicle (if gavage): stability measured at concentrations up to 250 mg/ml at room temperature.
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Remarks:
See Analytical Report FPBSTUDY-156-VAL1: Torok J, Validation of Analytical Method for the Determination of ZEF 6099/100 from Gavage Media. FumoPrep Ltd., H-1044 Budapest, Ipari park u. 10, Hungary.
Details on analytical verification of doses or concentrations:
Instrument: Near infrared spectrophotomer: MicroNIR™ Pro 1700 ES
Mode: Transmission
Range: 908 – 1676 nm
Integration time: 10.2 ms
Scan Count: 100
Cuvette: Quartz, 1.000 cm

Objective: multivariate evaluation 5-5 NIR spectra of the test item solution at 4.8 mg/mL concentration level. The spectra of the test solution and the vehicle were compared. The spectra were pre-treated with standard normal variate normalization. There was intensive signal of the vehicle. The principal component analysis of the spectral data matrix eliminated the variables that carried no variance, resulting in spectra are informative.

Sample size: 7
LOQ: 4.90 mg/mL (0.542 m/m%)

Recovery:
92.3% at 15 mg/ml test solution.
91.4 % at 60 mg/ml
106.1% at 200 mg/ml

Precision:
5.79 % at 15 mg/ml test solution.
1.21 % at 60 mg/ml
1.06% at 200 mg/ml

The accuracy of the calibration solution was between 98.5 and 102.6% with 2.32% RSD (n=3)

Duration of treatment / exposure:
28 days for males, up to 58 days for females
Frequency of treatment:
once daily, 7 days per week.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first dose, and at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at randomisation, before treatment, at day 0, weekly and at necropsy. Females also at GD0, 3, 10, 17 and 20 and at PPD 0 and 4.

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (pentobarital)
- Animals fasted: Yes, prior to sacrifice
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine sampling will be performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, on the control and high dose group animals.
Other examinations:
Functional Observation Battery on 5 animals/group.

Body and organ weight: At the time of termination, body weight and weight of the following organs of all adult animals will be determined:
uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, thyroids with parathyroids. Testes and epididymides will be weighed individually. Individual and/or paired absolute organ weight will be reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable will be summarised. Relative organ weight (to body and brain weight) will be calculated and reported.

The number of implantation sites and of corpora lutea will be recorded in the females as applicable.
Statistics:
Data is collected using software PROVANTIS v.9. Statistical analysis is performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups will be checked by Bartlett's test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant, then Duncan's Multiple Range test is used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data is examined by Kolmogorow-Smirnow test. In the case of non- normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance is applied. If a positive result is detected, the inter-group comparisons are performed using Mann-Whitney U-test. The Chi-squared test will be used for non-continuous data.
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree is applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups will be checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log- transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett (Multiple Range) test is used to assess the significance of inter- group differences; identifying differences of <0.05 or <0.01 as appropriate.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted which were associated with the test material. Four males in the low dose group displays topical scars (with or without wounds) due to fighting. Red eye discharge was observed in one high dose male. No signs were noted in female animals.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
A statisitically significant decrease in the amount of basophilic neutrophils was observed in high dose males, but this was within the normal historical range of values. There was no similar decrease in the high dose females. Therefore, this was not considered toxicologically significant.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
A statisitically significant decrease in total protein concentration was observed in low dose males, but the data from all males was within the normal historical range of values, and there was no dose response. It is not considered toxicologically relevant or related to treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Statisitically significant differences in pH of the urine were observed in low and mid dose males, but this was within the normal historical range of values; it was not dose related, and did not occur in females. It was considered unrelated to treatment.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Description (incidence and severity):
Discussed as behavioural findings using the Functional Observational Battery.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Several findings typical of background conditions found in this strain of rat were observed and considered incidental. This included: minimal cardiomyocytes degeneration in 2/5 males and 1/5 females, minimal mixed mononuclear infiltrate in the myocardium of 1/5 males, slight tubular degeneration of right kidney in 1/5 males, minimal tubular basophilia of right kidney in 1/5 males and minimal intraluminal mineralization of the right kidney (papilla) in 1/5 males, minimal focal hepatocellular necrosis in 1/5 males, and minimal multinucleated giant cells in the testes of 1/5 males.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
No test material related changes were noted in any parameter examined in this study.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
NOAEL > 1000 mg/kg bw/d
Key result
Critical effects observed:
no
Conclusions:
In an OECD 422 guideline study of ZEF 6099/100 in the Wistar rat at doses of 100, 300 and 1000 mg/kg bw/d, the test material did not result in adverse effects in clinical signs, body weight, organ weights, food consumption, neurological assessment, haematology, coagulation, clinical chemistry, urinalysis, or gross/histopathology. There were no effects in reproductive parameters nor in F1 offspring viability, clinical signs, development or macroscopic examination. The NOAEL is greater than 1000 mg/kg bw/d.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
{3-hydroxy-4-[(9Z)-octadec-9-enoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate; {3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate
EC Number:
917-830-2
Cas Number:
1186514-91-9
Molecular formula:
not available for UVCB substances
IUPAC Name:
{3-hydroxy-4-[(9Z)-octadec-9-enoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate; {3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexyl}methyl 3-hydroxy-4-[(9Z,12Z)-octadeca-9,12-dienoyloxy]cyclohexane-1-carboxylate
Test material form:
liquid
Details on test material:
Lot # 210164, brown liquid, homogeneous, Expiry date: 2017-12-16

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation:369-460 g (males); 225-288 (females)
- Fasting period before study: no, fasting only before sacrifice
- Housing: males housed in groups of 4, in polycarbonate cages. Females housed singly during and after mating. Lignocell (R) and Arbocel (R) natural crinklets
- Diet (e.g. ad libitum): at lib, ssniff (R)
- Water (e.g. ad libitum): ad lib, municipal water supply
- Acclimation period: at least 5 days
DETAILS OF FOOD AND WATER QUALITY: ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 29 Nov 2016 To: 13 Jan 2017.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate vehicle for oily liquid test material
- Concentration in vehicle: 100, 300 and 1000 mg/kg bw/d
- Amount of vehicle (if gavage): stability measured at concentrations up to 250 mg/ml at room temperature.
- Lot/batch no. (if required): BCBQ0052V, Sigma-Aldrich
- Purity:
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation or up to 14 days
- Proof of pregnancy:vaginal plug or sperm in vaginal smear/ referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes, for up to 7 days
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Remarks:
See Analytical Report FPBSTUDY-156-VAL1: Torok J, Validation of Analytical Method for the Determination of ZEF 6099/100 from Gavage Media. FumoPrep Ltd., H-1044 Budapest, Ipari park u. 10, Hungary.
Details on analytical verification of doses or concentrations:
Instrument: Near infrared spectrophotomer: MicroNIR™ Pro 1700 ES
Mode: Transmission
Range: 908 – 1676 nm
Integration time: 10.2 ms
Scan Count: 100
Cuvette: Quartz, 1.000 cm

Objective: multivariate evaluation 5-5 NIR spectra of the test item solution at 4.8 mg/mL concentration level. The spectra of the test solution and the vehicle were compared. The spectra were pre-treated with standard normal variate normalization. There was intensive signal of the vehicle. The principal component analysis of the spectral data matrix eliminated the variables that carried no variance, resulting in spectra are informative.

Sample size: 7
LOQ: 4.90 mg/mL (0.542 m/m%)

Recovery:
92.3% at 15 mg/ml test solution.
91.4 % at 60 mg/ml
106.1% at 200 mg/ml

Precision:
5.79 % at 15 mg/ml test solution.
1.21 % at 60 mg/ml
1.06% at 200 mg/ml

The accuracy of the calibration solution was between 98.5 and 102.6% with 2.32% RSD (n=3)

Duration of treatment / exposure:
28 days (males), up to 44 days (females)
Frequency of treatment:
daily
Details on study schedule:
Dosing of Male Animals:
Acclimatisation At least 5 days
Pre-mating period 14 days
Mating/Post-mating period at least 14 days

Last week of treatment: FOB (e.g. Day 25 am, 5 animals/group)
Prior to/at necropsy: Urinalysis, i.e. sampling start on Day 27, end Day 28 after approximately 16 hours (5 animals/group). Coagulation evaluation, Clinical chemistry and Haematology (5 animals/group). Necropsy with organ weight determination (i.e. Day 28 or later, all males).

Females:
Acclimatisation period: at least 5 days
Pre-mating period: 14 days
Mating: Up to 14 days
Gestation: 22-24 days
Delivery: 1 day
Lactation period: at least 4 days: FOB (5 animals/group) on PND4, Pups necropsy on PND 4. Dams fasted overnight prior to necropsy.
PND5: Prior to/at necropsy:
- Urinalysis (i.e., sampling start on PND 4, end PND5 after approximately 16 h, 5 animals/group).
- Coagulation evaluation, Clinical chemistry and Haematology (5 animals/group)
- Necropsy with organ weight determination (i.e. all females)



Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first dose, and at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at randomisation, before treatment, at day 0, weekly and at necr opsy. Females also at GD0, 3, 10, 17 and 20 and at PPD 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE: not a feeding study
WATER CONSUMPTION AND COMPOUND INTAKE: not a drinking water study

OTHER:
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (pentobarital)
- Animals fasted: Yes, prior to sacrifice
- How many animals:
- Parameters checked in table [No.?] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:
- Parameters checked in table [No.?] were examined.
URINALYSIS: Yes


- Time schedule for collection of urine: Urine sampling will be performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
OTHER:
Sacrifice and pathology
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, on the control and high dose group animals.
Other examinations
Functional Observation Battery on 5 animals/group.

Body and organ weight: At the time of termination, body weight and weight of the following organs of all adult animals will be determined:
uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, thyroids with parathyroids. Testes and epididymides will be weighed individually. Individual and/or paired absolute organ weight will be reported for each animal and adjusted for the body and brain weights. Paired organ weights as ap plicable will be summarised. Relative organ weight (to body and brain weight) will be calculated and reported.
Oestrous cyclicity (parental animals):
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non mated females
- Duration of pregnancy (days)
- Number of Corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups Day 0 and 4
- * Pre-implantation mortality
- * Intrauterine mortality
- * Total mortality (intra and extra uterine mortality)
Detailed histological examination of the ovaries will cover the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Sperm parameters (parental animals):
- Number of pairings
- Number of fertile pairings
- Number of infertile males
Special attention will be paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Females will be allowed to litter and rear their offspring. Delivery process will be observed as carefully as possible. Dams will be observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling will be observed by the presence of milk in the pups' stomach. Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), the presence of gross abnormalities and any abnormal behaviour of the offspring. Live pups will be counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-partum, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters will be checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) will be subjected to necropsy with macroscopic examination and the cause of death will be identified if possible.
Postmortem examinations (parental animals):
Gross necropsy will be performed on each animal. Terminally (one day after the last treatment), animals will be sacrificed under anaesthesia by exsanguination; anaesthetic product may be diluted for pups’ euthanasia as required.
For the adult animals, detailed histological examination will be performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• any animals found dead or euthanized pre-terminally during the study in all groups
• all macroscopic findings (abnormalities), except of minor order from all animals
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and of all males that failed to sire and all females that failed to deliver healthy pups
Postmortem examinations (offspring):
- Mean pup body weight (per pup within the group and per litter) on PND 0 and 4
- Mean pup body weight gain (per litter) between postnatal Days 0-4
The pups found dead and intact (not cannibalized) will be subjected to necropsy with macroscopic examination and the cause of death will be identified if possible. All observed abnormalities will be recorded.
Statistics:
Data is collected using software PROVANTIS v.9. Statistical analysis is performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups will be checked by Bartlett's test. Where no significant heterogeneity is detected, a one-way analysis of vari ance (ANOVA) is carried out. If the obtained result is significant, then Duncan's Multiple Range test is used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data is examined by Kolmogorow-Smirnow test. In the case of non- normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance is applied. If a positive result is detected, the inter-group comparisons are performed using Mann-Whitney U-test. The Chi-squared test will be used for non-continuous data.
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree is applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups will be checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log- transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett (Multiple Range) test is used to assess the significance of inter- group differences; identifying differences of <0.05 or <0.01 as appropriate.
Reproductive indices:
* Female mating index
* Female fertility index
* Gestation index
* Male mating index
* Male fertility index
Offspring viability indices:
* Survival Index of pups on postnatal Days 0 and 4
* Sex ratio % (on postnatal Days 0 and 4)
Number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Low dose males showed decreased basophillic neutrophil concentrations. Not treatment related/toxicologically significant
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Low dose males showed decreased protein concentrations. Not treatment related/toxicologically significant
Urinalysis findings:
no effects observed
Description (incidence and severity):
Low dose males showed altered urinary pH. Not treatment related/toxicologically significant
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating and fertility indices were 100% for both males and females. Successful matings occurred within 3 days for all dose groups. The gestation index was 100% for all groups other than low dose females, which showed a gestation index of 92%.

Details on results (P0)

The number of corpora lutea and number of implantation sites were comparable between all dose groups and the controls.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
NOAEL > 1000 mg/kg bw/d

Results: P1 (second parental generation)

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were 3 Control, 1 Low Dose, 4 Mid Dose and 1 High dose pups found dead. The cannibalization of 1, 2, 4 and 1 pups from the Control, Low, Mid and High dose groups respectively was observed at necropsy.
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:
not examined
Description (incidence and severity):
No test item-related macroscopic findings were observed in the F1 generation in any dose group (PN0-4).
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The number of viable pups on PND 0 and PND 4 as well as pups’ survival indices on PND 0 and PND 4 were comparable to control values at up to and including 1000 mg/kg bw/d. The number of pups died during the postnatal period in the test item treated groups was comparable with control data.

The sex ratio of female pups was lower (not statistically significant) in all test item treated groups (both on PND0 and PND4) when compared to the control data, therefore it was not considered as a test item effect.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The NOAEL is greater than 1000 mg/kg bw/d.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
In an OECD 422 guideline study on ZEF 6099/100 at doses of 100, 300 and 1000 mg/kg bw/d in Wistar rats, no adverse effects were observed in fertility, mating, gestation or development of the offspring. No teratological effects were observed. The NOAEL is greater than 1000 mg/kg bw/d.