Fatty acids, tall-oil, reaction products with linseed oil, maleic anhydride and methanol

Toxicity to reproduction

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: Males: 360-392 g, Females: 228-261 g
- 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:

polyethylene glycol

Remarks:

PEG 400

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): BCBQ0052V, Sigma-Aldrich

Analytical verification of doses or concentrations:

yes

Remarks:

See Analytical Report #16-178-316AN. Sarvari, Z., 22 March 2017. Analysis by LC-MS. Stable in PEG 400 for 16 days at room temperature (104% recovery). Stock solution stable for 7 days at 5 degrees C.

Details on analytical verification of doses or concentrations:

Analysed by LC-MS.
HPLC-MS: Thermo-Finnigan Surveyor HPLC with LCQ Duo MS detector
Balance: Sartorius BP221S
Water purification system: MILLIPORE, DIRECT Q 8 UV

Analytical Technique: LC-MS
Column: Luna CN, 50×4.6 mm, 3 µm
Column temperature: 25 °C
Mobile Phase: Methanol : water = 8:2 +0.1% acetic acid
Flow: 0.5 mL/min
Detector: SIM (409.1 m/z, negative ion)

Linearity test 1: Y = -50958.8+286873*X+8325.51*X^2 R^2 = 0.9993 W: Equal.
Linearity test 2:
Y = -689226+836889*X R^2 = 0.9955 W: 1/X

Recovery and Precision of Analytical test: 93-104%
Stabillity of Test Item in Vehicle: 104-109%

Duration of treatment / exposure:

28 days for males, 34-44 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:

5

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.

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.

Name: Euthanimal® (40% Pentobarbital sodium)
Batch No.: 1409236-06
Expiry Date: September 2017
Produced by: Alfasan Netherland BW, Kuipersweg 9, Woerden
Storage: Room temperature
Purpose of use: Euthanasia

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

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control group male was found dead on day 17, due to technical error (perforation of the esophagus, confirmed 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:

effects observed, treatment-related

Description (incidence and severity):

Mean prothrombin time was significantly decreased (p<0.05) in mid and high dose males, but the treat ed group values were in the normal control range, and there was no clear dose response. A similar trend was noted in females, but didn't reach statistical significance. These effects are not considered biologically relevant or a test item related effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Glucose concentration was significantly increased (p<0.01) in mid and high dose males, but the treat ed group values were in the normal control range. These effects are not considered toxicologically significant or related to treatment.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weight (absolute and relative) was significantly increased (p,0.05) in both sexes at the high dose. For males (but not females) in the high dose group, this liver weight was just above the historical control range. Liver microscopic appearance and liver function tests remained unaffected. These effects are not considered a test item related adverse effect. There were significant decreases in the absolute weight of brain and epididymus for all test item related groups (males). Absolute weight of spleen was decreased significantly in high dose males, and that of the thumus in mid dose males. These were within the historical control range, with no evidence of histopathology. These were not considered a test item related adverse effect.

Neuropathological findings:

no effects observed

Description (incidence and severity):

.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

All high dose males showed a minimal increase in number of eosinophilic droplets in the kidney, compared with normal background presence in control group males. These cytoplasmic/luminal droplets had similar size to those noted in controls. Droplets were predominantly located in the proximal tubul es of the cortex. No accompanying degenerative/necrotic and inflammatory changes were recorded.
In the mid dose males, there was reduced sperm content and an occurrence of spermatocele in the epididymis (1/5) and dilation of renal tubule (1/5). In control males, there was inflammation of the prostate gland (3/5) this also occurred in 2/5 high dose males. In one high dose female, congestion/ haemorrhage of the thumus occurred.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant toxicity observed at the highest dose administered.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

ANALYSIS OF DOSE FORMULATIONS:

The measured concentrations of ZWA 5496/100 evaluated for each dose group varied between 93% and 106% of the nominal value. No test item was detected in the control samples at any occasion. These results were within the acceptable range (90% - 110% of the nominal concentration) and were considered to be suitable for the study purposes.

 

All test item formulation samples were found to be homogeneous. Formulations were considered to be adequately stable under the study conditions.

Conclusions:

In an OECD 422 guideline study (28-day repeated dose toxicity and reproductive toxicity screening study) in Wistar rats at doses of the test material of 100, 300 and 1000 mg/kg bw/day, there were no adverse effects noted. There were minor changes in organ weights of liver and kidney of both sexes, and in males for epididymus, brain and spleen, but these were not associated with impaired function or histopathology findings. There was no reproductive toxicity observed. The conclusion is that the substance does not exert toxic effects at any dose tested under these conditions. The NOAEL for repeated dose toxicity effects is 1000 mg/kg bw/day.

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

Justification for study design:

Annex VIII requirement of Regulation EC No. 1907/2006

Test material

Test material information

Constituent 1

Reference substance name:

Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol

EC Number:

926-195-0

Cas Number:

1176284-65-3

Molecular formula:

not available for the UVCB substance

IUPAC Name:

Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol

Test material form:

liquid

Details on test material:

UVCB, 100% purity, Batch no. 210150084, brown liquid.

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: Males: 360-392 g, Females: 228-261 g
- 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:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations may be prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results of the analytical method validation study
- Mixing appropriate amounts with (Type of food): no
- Storage temperature of food:

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

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 #16-178-316AN. Sarvari, Z., 22 March 2017. Analysis by LC-MS. Stable in PEG 400 for 16 days at room temperature (104% recovery). Stock solution stable for 7 days at 5 degrees C.

Details on analytical verification of doses or concentrations:

Analysed by LC-MS.
HPLC-MS: Thermo-Finnigan Surveyor HPLC with LCQ Duo MS detector Balance: Sartorius BP221S
Water purification system: MILLIPORE, DIRECT Q 8 UV

Analytical Technique: LC-MS Column: Luna CN, 50×4.6 mm, 3 µm Column temperature: 25 °C
Mobile Phase: Methanol : water = 8:2 +0.1% acetic acid Flow: 0.5 mL/min
Detector: SIM (409.1 m/z, negative ion)

Linearity test 1: Y = -50958.8+286873*X+8325.51*X^2
R^2 = 0.9993 W: Equal. Linearity test 2:
Y = -689226+836889*X R^2 = 0.9955 W: 1/X

Recovery and Precision of Analytical test: 93-104% Stabillity of Test Item in Vehicle: 104-109%

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 / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

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 necropsy. 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

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- norma
l 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 dec ision 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:

mortality observed, non-treatment-related

Description (incidence):

One control group male was found dead on day 17, due to technical error (perforation of the esophagus, confirmed at necropsy).

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:

effects observed, treatment-related

Description (incidence and severity):

Mean prothrombin time was significantly decreased (p<0.05) in mid and high dose males, but the treated group values were in the normal control range, and there was no clear dose response. A similar trend was noted in females, but didn't reach statistical significance. These effects are not considered a test item related adverse effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Glucose concentration was significantly increased (p<0.01) in mid and high dose males, but the treated group values were in the normal control range. These effects are not considered a test item related adverse effect.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Total urine volume in the high dose group was much smaller than that of controls, but the difference was not statistically significant. These effects are not considered a test item related adverse effect.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

All high dose males showed a minimal increase in number of eosinophilic droplets in the kidney, compared with normal background presence in control group males. These cytoplasmic/luminal droplets had similar size to those noted in controls. Droplets were predominantly located in the proximal tubules of the cortex. No accompanied degenerative/necrotic and inflammatory changes were recorded. In the mid dose males, there was reduced sperm content and an occurrence of spermatocele in the epididymis (1/5) and dilation of renal tubule (1/5). In control males, there was inflammation of the prostate gland (3/5) this also occurred in 2/5 high dose males. In one high dose female, congestion/haemorrhage of the thumus occurred.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

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):

One low dose male failed to sire, and one female did not become pregnant. No test item-related changes were observed in those animals.

Effect levels (P0)

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:

No adverse effects attributable to the test material were observed

Target system / organ toxicity (P0)

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:

no mortality observed

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:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related gross findings were observed in the F1 generation in any dose group (PN0-4).

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

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

gross pathology

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No adverse effects attributable to the test material were observed.

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)

Any other information on results incl. tables

ANALYSIS OF DOSE FORMULATIONS:

The measured concentrations of ZWA 5496/100 evaluated for each test item containing dose group varied between 93% and 106% of the nominal value. No test item was detected in the control samples at any occasion. These results were within the acceptable range (90% - 110% of the nominal concentration) and were considered to be suitable for the study purposes.

 

All test item formulation samples were found to be homogeneous. Formulations were considered to be adequately stable under the study conditions.

REPRODUCTIVE PARAMATERS:

Table 4: Summary of reproductive parameters (males)

Parameters	Group /Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	12
Number of pre-terminal death	1	0	0	0
Number of males used for mating	12	12	12	12
Number of successful mating	12	12	12	12
Number of infertile animals	0	1	0	0
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	92	100	100

Note: One male in the Control group was found dead on Day 17, but as it happened after a successful mating on Day 14, it was included in the calculation.

 

Table 5: Summary of reproductive parameters (females)

Parameters	Group /Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	12
Number of pre-terminal death	0	0	0	0
Number of females used for mating	12	12	12	12
Number of sperm positive females	12	12	12	12
Number of females with no implantation sites	0	1	0	0
Number of pregnant females	12	11	12	12
Number of pregnant females with live born(s)	12	11	12	12
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	92	100	100
Female gestation index (%)	100	100	100	100

Note: One female in the Low dose group (#2512) was not-pregnant.

Table 6: Summary of the intrauterine evaluation

Parameters	Group /Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of evaluated females	12	11	12	12
Mean number of corpora lutea	16.6	14.7	15.2	16.5	NS
Pre-implantation mortality, mean	1.0	0.0	0.3	0.6	NS
Pre-implantation mortality (%), mean	6.49	0.00*	1.66	3.44	DU
Mean number of implantations	15.6	14.7	14.9	15.9	NS
Intrauterine mortality, mean	0.9	0.8	0.8	0.9	NS
Intrauterine mortality (%), mean	6.64	5.16	5.33	6.01	NS
Number of pups born, mean	15.4	14.2	14.5	15.7	NS
Number of live born pups, mean	14.7	13.9	14.2	15.0	NS
Post-natal mortality, mean	0.0	0.0	0.0	0.0	NS
Post-natal mortality (%), mean	0.00	0.00	0.00	0.00	NS
Total mortality, mean	1.1	0.8	0.8	1.2	NS
Total mortality (%), mean	7.82	5.81	5.97	7.37	NS
Duration of pregnancy (days)	21.92	21.86	21.92	21.58	NS

Notes: Mean values were rounded to one or two decimal places.

* = p<0.05, ** = p<0.01

DU: Dunn test, NS: Statistically not significant

Applicant's summary and conclusion

Conclusions:

In an OECD 422 guideline study (28-day repeated dose toxicity and reproductive toxicity screening study) in Wistar rats at doses of the test material of 100, 300 and 1000 mg/kg bw/day, there were no test item related adverse effects noted. There were no effects on reproductive indices, and there were no teratogenic effects in the offspring. The conclusion is that the substance does not exert reproductive toxicity effects at any dose tested under these conditions. The parental NOAEL and the NOAEL for offspring are both greater than 1000 mg/kg bw/day.