Fatty acids, C16-18 and C18-unsatd., reaction products with glycidyl neodecanoate and 7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects observed in an OECD 422 guideline study on ZEF 6099/100 at doses up to 1000 mg/kg bw/d.

Link to relevant study records

Reference

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

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)

Limit test:

no

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.

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)

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

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

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: 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%.

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

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

adequate

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No adverse effects were observed in an OECD 422 guideline study in Wistar rats, either in the P0 (parental) or F1(offspring) generations (to PND4), on ZEF 6099/100 at doses up to 1000 mg/kg bw/d. There were no teratological or reproductive effects found.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

In a repeated dose oral toxicity and reproductive toxicity screening study in the Wistar rat for 28 days according to the OECD 422 method, no significant reproductive toxicity was observed. There was no evidence of developmental toxicity in the offspring of this study. The NOAEL for reproduction is greater than the highest dose tested, 1000 mg/kg bw/day.

Mode of Action Analysis / Human Relevance Framework

Not applicable

Justification for classification or non-classification

In a repeated oral (gavage) dose toxicity and reproductive toxicity screening study in the rat for 28 days according to the OECD 422 method, no significant reproductive/developmental toxicity was observed. The NOAEL for reproduction is higher than the highest dose tested, 1000 mg/kg bw/day. The criteria for Regulation EC No. 1272/2008 for classification for reproductive toxicity are not met. The substance is not classified.