Benzene, mono-C11-13-branched alkyl derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In life phase: 02 February 2021 - 12 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzene, mono-C11-C13-branched alkyl derivatives

Cas Number:

2156592-70-8

Molecular formula:

Main component may be represented by formula C18H30

IUPAC Name:

Benzene, mono-C11-C13-branched alkyl derivatives

Test material form:

liquid: viscous

Remarks:

Fluid, oily colourless substance

Details on test material:

Batch number 1090914
Expiry date: 01 September 2022

Specific details on test material used for the study:

- Source of test material: Manufacturer
- Purity: 100% (UVCB)
- Substance as produced (no purification, treatmennt, separation, etc.)
- Storage condition: at controlled room temperature (15-25℃, ≤70% relative humidity), protected from humidity and from light (tight closed container).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The test system and the number of animals used in the study are in compliance with the relevant OECD No. 421 [1] guideline. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. Sprague Dawley rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.
The minimum number of animals will be used, corresponding to the regulatory guidelines being followed, but taking into consideration the scientific reliability of the collected information.

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, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany)

- Hygienic level: SPF at arrival, standard laboratory conditions during the study Number of groups: 5 groups; one control and 4 test item-treated groups

- Number/sex of animals:
63 male and 66 female rats were used in the acclimatisation/pre-treatment period.
60 male and 60 female rats (12/sex/group) were used for treatment
Animals were originated from different units, to avoid brother/sister mating.

- Age of animals: Young adult rats, 11 weeks old at starting and 13 weeks at mating.

- Females (if applicable) nulliparous and non-pregnant: yes

- Body weight: Males: 399-489 g, females: 223-307 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.

- Animal Screening: The health status of the animals assigned to study was verified by the clinical Veterinarian. Females will be nulliparous and non-pregnant.

- Environmental Acclimation: Environmental acclimation period for the study was 19 days.

-Selection, Assignment and Disposition of Animals
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that animals of all test groups are as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
Any unused, spare animals were moved back to the stock colony in the end of the study.

- Housing:
Animal room: 523
Cage type: Type II and/or III polycarbonate
Bedding / nesting: SAFE 3/4-S Hygienic Animal Bedding (Batch numbers: 03027201024 / 03027201208, Expiry dates: 24 October 2023 / 08 December 2023) and SAFE Crinklets Natural material (Batch number: 05072200824, Expiry date: 24 August 2023) (nesting material) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany), were used in the study. Details of the bedding and nesting materials were included in the raw data binder and archived.
Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired* or individually housed (with pups), respectively.
*Note: Males were individually housed after their mating finished until the end of the mating period

Animal Enrichment:
Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities. Nesting material allowed normal nesting behaviour. Certified cardboard hiding tubes (GLP Mini Fun Tunnels, Batch number: A/123 LBS (Serving Biotechnology) Ltd, Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals, details of the quality were archived with the raw data.
 
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch number: 713 70882, Expiry date: 30 April 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum. The supplier provided an analytical certificate for the batch used, which included in the raw data binder and archived.
The food was routinely analysed and was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

- Water (e.g. ad libitum): Animals received tap water from the municipal supply, as for human consumption from a 500
mL bottle, ad libitum.
Quality control analysis of the water was performed at least once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (Address: H-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are included in the raw data and are archived at the Test Facility.
The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
 
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-25.9℃ (target range: 19-25℃)
- Humidity (%): 29-65% (target range: 30-70%)
- Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Ventilation: 15-20 air exchanges/hour

IN-LIFE DATES: From: 02 February 2021 To:12 April 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Propylene glycol + 1% Polysorbate

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
----Preparation of Formulations
As agreed with the Sponsor, no correction for purity of the test item was applied during formulation.
The test item was formulated in the selected vehicle (Propylene glycol + 1% Polysorbate) as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of the Test Facility.
Formulations were prepared fresh every day prior to administration to animals.
--- Preparation Details
The calculated amount of the test item was weighed into a clean, calibrated glass container and then mixed properly (with magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation.


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
Based on the available information provided by the Sponsor as well as results of a trial formulation Propylene glycol + 1% Polysorbate was selected as vehicle for this study in agreement with the Sponsor. Propylene glycol + 1% Polysorbate was considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.

The relevant data of the chemicals used for vehicle of the study are shown below. The Certificates of Analysis of the vehicle are included in the study report (Appendix 2).

Details of the used vehicle see below:
Name: Propylene glycol + 1% Polysorbate
Dispense codes: S51017 / S51018 / S51019 / S51020 / S51021 / S51022 / S51023
Expiry Dates: 12 March 2021 / 20 March 2021 / 26 March 2021 / 08 April 2021 / / 15 April 2021 / / 26 April 2021 / / 09 May 2021
Storage condition: Room temperature

Components of the vehicle used in the study:

Name (Synonym): 1,2-Propanediol (Propylene glycol)
Manufacturer: ThermoFisher Scientific
Batch number: 1920944
Expiry dates: 31 March 2023 / 31 May 2023
Storage conditions: Room temperature

Name (Synonym): Polysorbate 80 (Tween 80)
Manufacturer: Sigma-Aldrich
Batch numbers: BCBV5152 / BCCB7399
Expiry dates: 30 June 2022 / 31 May 2024
Storage conditions: Room temperature

Amount of vehicle:
The control group was treated with the vehicle only (Propylene glycol + 1% Polysorbate) at the dose volume of 5 mL/kg bw.
The test item treated groups received the same dose volume (5 mL/kg bw with a dose level of test item ranging from 12.5 mg/kg bw/day to 800 mg/kg bw/day).


ADMINISTRATION OF TEST ITEM

The dosing solutions were administered to the test item or vehicle-treated (control) animals daily, in the morning, 7 days/week, by oral gavage, using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

Dosing of both sexes began after the acclimatisation (5 days) and pre-exposure period (14 more days), and treatment was performed for 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.

The first day of dosing of each animal was regarded as Day 0.

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.

------Dosing scheme, Male animals:
-- Acclimatisation-(5 days),-Pre-exposure period (14 days)
-- Pre-mating period 14 days
-- Mating/Post-mating period 14 days: Prior to/at necropsy: Sampling for potential thyroid hormone analysis and Necropsy with organ weight determination (Day 28, all males)


Females were dosed for 14 days pre-mating, for up to 6 days mating period, through gestation and up to and including the day before necropsy (at least 13 days post-partum dosing). The day of birth (when parturition is complete) was defined as Day 0 post-partum. Females not delivered were sacrificed as practical (on Day 50).

---Dosing scheme, Female animals:
-- Acclimatisation (5 days), Pre-exposure period (14 days): Oestrus cycle monitoring during the pre-exposure period
-- Pre-: mating period 14 days: Oestrus cycle monitoring
-- Mating Up to 6 days: Oestrus cycle monitoring
-- Gestation ~22-23 days: -
-- Delivery
-- Lactation periodat lea st 13 days: Pups necropsy (i.e. PND13)
-- PND14: Prior to/at necropsy:
- Sampling for potential thyroid hormone analysis
- Necropsy with organ weight determination (i.e. all females)


All selected F1 offspring were terminated on Post-natal Day (PND) 13 (F1 offspring selected for blood sampling were terminated on PND4 due to technical reasons, to be documented and reported). In order to allow the overnight fasting of dams prior necropsy on PPD14, offspring was euthanized on PPD/PND13, and the dams on PPD/PND14.

Details on mating procedure:

Sixty-three male and sixty-six female Sprague Dawley rats were used in the pre-treatment period. At the end of the pre-treatment period, only 60 females majority of them showing regular oestrus cycles and 60 males were allocated to treatment groups (12 animals/sex/groups, 5 groups). Animals were assigned to groups before the start of the treatment as follows:

Group No. Group Designation Dose level (mg/kg bw/day) Concentration of formulation (mg/mL) Dose volume (mL/kg bw) Animal numbers
Males Females
1 Control - - 5 12 12
2 Low Dose 12.5 2.5 5 12 12
3 Mid Dose 50 10 5 12 12
4 Dose 200 40 5 12 12
5 Ultra-High Dose 800 160 5 12 12

The control group was treated with the vehicle only (Propylene glycol + 1% Polysorbate) at the dose volume of 5 mL/kg bw.
Any spare animals were moved to the spare colony of the Test Facility.

Mating procedure
Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurs, for up to 6 days. A vaginal smear was prepared daily during the mating period and stained with 1% (w/v) aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item was formulated in the selected vehile (Propylene glycol+ 1% Polysorbate) as a visible stable homogenous solution.

Concentration and homogeneity of the dose formulations were determined three times during the study. On each sampling occasion, top, middle and bottom duplicate samples were taken from test item formulations for concentration and homogeneity measurement, one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
Formulations were prepared daily. No test item was detected in the negative (vehicle) control sample.

Additional sample was collected for confirmatory purposes in case of the Mid dose formulation

After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.

All test item formulations were shown to be homogeneous and they were found to be in the range of 93 to 106% of nominal concentrations.
Based on these results, the formulations were considered suitable for the study purposes.


See table 1 Analytical Results in "any other information on materials and methods incl. tables" chapter

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 6 days mating period, through gestation and up to and including the day before necropsy (at least 13 days post-partum dosing). The day of birth (when parturition is complete) was defined as Day 0 post-partum.

Frequency of treatment:

Daily in the morning, 7 days/week

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

12.5 mg/kg bw/day

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day

Remarks:

Mid Dose

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day

Remarks:

High Dose

Doses / concentrations 5

Dose / conc.:

800 mg/kg bw/day

Remarks:

Ultra-High Dose

No. of animals per sex per dose:

12 Males
12 Females

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of an OECD 407 study (CIT/Study No. 36887 TSR) and 90 days toxicity test (FR - 11995.307.001.13), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.

In the 28 day study, hypersalivation was recorded with slightly lower body weights, with clearly adverse effects on clinical pathology in the 800 and 400 mg/kg bw/day dose groups. Macroscopic findings were observed in the liver, adrenals, prostate, seminal vesicles, ovaries, thymus, spleen. These changes correlated with treatment-related microscopic observations in the liver (hepatocellular hypertrophy), forestomach (hyperplasia and hyperkeratosis), adrenals (cortical hypertrophy) and kidneys (tubular mineralization in males). Atrophic changes were seen in the prostate, seminal vesicles, testes and thymus (males only).

In the 90 days toxicity test the test item was administered in diet at dose levels of 300, 100 and 3000 ppm. There were test item related effects on food consumption, body weight and body weight gain in the High dose group. Microscopic changes were observed in testis and epididymis in treated males, in liver in females and in kidneys, thymus and spleen in both sexes. Statistically significant higher frequencies of all types of spermatozoid abnormalities were observed in the High dose group. No NOAEL could be identified because significant adverse effects were observed on sperm at 300 ppm.

Based on these data, 800 mg/kg bw/day was chosen as High dose level in this study. Lower doses were spaced with a factor of 4.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

- Mortality/Morbidity Checks
Animals were inspected for signs of morbidity and mortality twice per day during the treatment (at the beginning and end of each working day) and once a day before the treatment period.
The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 was taken into consideration [5].

- Clinical Observations
Any clinical sign noted during dosing or at any other occasions was recorded at the time seen.

- Cage Side Observations
General clinical observations were made once a day*, during the pre-treatment and treatment period in the afternoon (pm).
*Note: No general clinical observations were made on the day of necropsy.

- Detailed Clinical Observations
Detailed clinical observations were made before the first day of treatment and then once a week. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality was recorded including onset, degree and duration of signs as applicable.
On Gestation Day 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

- Body Weights
All male animals were weighed with accuracy of 1 g on the first day of dosing then once a week and at termination or death.
All female animals were weighed with accuracy of 1 g on the first day of dosing then once a week until mating. Parent females were weighed on Gestation Day (GD) 0, 3, 7, 14, 17 and 20, on PPD (Post-partum Day) 0, 4, 7, 10 and 13, and at termination.
Non-pregnant females were weighed with accuracy of 1 g at least once a week until sacrifice.

- Food Consumption
Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g weekly until the start of the pairing period (on a body weight measurements day). No food consumption was measured during mating. Food consumption was measured on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0, 4, 7, 10 and 13.
Main daily food consumption was calculated for each interval.

- Water Consumption
No water consumption was measured in the study.

- Ophthalmic Examinations
No ophthalmoscopy was measured in the study.

Oestrous cyclicity (parental animals):

Oestrus cycles was monitored by vaginal smears daily during pre-dosing (12 females/group for 2 weeks) and also checking daily from the beginning of the treatment period until evidence of mating.

Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurs, for up to 6 days. A vaginal smear was prepared daily during the mating period and stained with 1% (w/v) aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually.

Additionally, vaginal smears were prepared and examined for each surviving female on the day of necropsy (PPD14) to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs (if necessary).

PRE-EXPOSURE PERIOD:
Each female selected for the study showed acceptable cycles (mean cycle length of 3.92-4.15 days was observed in the different groups) before starting the treatment period

EXPOSURE PERIOD (pre-mating and mating periods):

No indication of test item related effect was seen in the oestrus cycle data during the pre-mating and mating periods (mean cycle length was 4.00, 4.03, 4.08, 4.13 and 4.23 days in the Control, Low, Mid, High and Ultra-High dose groups, respectively).

Prolonged oestrus was recorded for one (1/12) High and three (3/12) Ultra-High dose females. The incidence in the Ultra High dose was considered as being a test item related effect.

Prolonged dioestrus was noted for one Low dose female, one Mid dose female and four Ultra High dose females.

Pseudo-pregnancy was noted for 1, 2, 3 and 2 in Low, Mid, High and Ultra-High dose females.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, levator ani plus bulbocavernosus muscle complex weight, Cowper's glands and glans penis weight.
daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Litter observations:

Observation of the delivery process, offspring and nursing instinct
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy.

Dams were observed to record whether they form a nest from the bedding material provided and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

Each litter was 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) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4 and PND 13, with accuracy of 0.01 g.

All the litters were checked and recorded daily for the number of viable, dead / autolysed and cannibalized pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) was also recorded on each day. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

On PND 4, litters were culled to yield, as nearly as possible, 5 males and 5 females per litter. Pups to be culled within each litter was selected randomly. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups are retained. Culling was not performed on litter sizes less than 10. All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.

The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0). The anogenital distance was also normalized to a measure of pup size (the cube root of body weight) for statistical analysis if considered appropriate by the Study Director.

One male and one female pup per litter (if possible) were previously selected for culling for blood sampling on PND 4.

Number of nipples/areolae in male pups was recorded on PND 13 (individual records was maintained).

All pups were necropsied on PND 13. Pups were examined for gross lesions (particular attention paid to reproductive development, genitals).

Postmortem examinations (parental animals):

SACRIFICE:

Females were allowed to litter and rear their offspring and were euthanized at study termination.

GROSS NECROPSY:

Gross necropsy was performed on each adult animal irrespective of the date of death. Terminally (males were euthanized after 28 days of dose administration, females on PND14, unmated females or non-delivered females was euthanised when the first dam sacrificed), animals were sacrificed under anaesthesia by exsanguination; anaesthetic product was diluted for pups’ euthanasia as required.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened and the appearance of the tissues and organs (special attention: reproductive system) were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle.
The number of implantation sites and of visible corpora lutea was recorded in the females.
Uteri of apparently non-pregnant rats was examined to confirm absence of implantation sites.


For animals found dead, no organ weight measurement was performed, but tissue or organ samples were retained for those animals

For euthanized animals, Weight of selected organs were measured, and selected organs and tissues were retained.


ORGAN WEIGHTS

At the time of termination, body weight and weight of the following organs of all adults males was determined:

• With a precision of 0.01 g: testes, epididymides, levator ani plus bulbocavernosus muscle complex
• With a precision of 0.001 g: Cowper’s glands and glans penis.
Testes and epididymides were weighed individually (paired organ weights as applicable were summarised). Relative organ weight (to body and brain weight) was calculated and reported. Individual and/or paired absolute organ weight were reported for each animal and relative organ weight (to body and brain weight) were calculated and reported. The weight of the brain was measured and the organ was preserved.

TISSUE COLLECTION AND PRESERVATION:

The weighed organs and all organs showing macroscopic lesions of all adult and all pups were preserved (including tissues from equivalent control group animals). The testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

For all the males, testes, epididymides, accessory sex organs and gross lesions were retained.

For all the females, ovaries, accessory sex organs and gross lesions (including evident target organs) were retained.

BLOOD SAMPLING for Thyroid Hormone Analysis:

For potential thyroid hormone analysis, blood samples were taken by venepuncture using vena sublingualis into tubes containing K3-EDTA as anticoagulant as follows:

• from all dams and at least two pups per litter on PPD 14 (females) / PND13 (pups),
• from eight non-pregnant adult females at termination,
• from all adult males at termination.

The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. Timing are documented in the raw data.
Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4°C). The resulting plasma was divided in at least two aliquots (volume target was at least 150 μL for the first aliquot and at least 150 µL for the second aliquot, any remaining sample (if any) was kept as a third aliquot) and stored in an ultra-freezer (-80±10°C) until analysis.
Thyroid hormone analysis was not required by the Sponsor’s Representative.

HISTOLOGY and MICROSCOPIC EVALUATION

No histopathology evaluation was performed.
If no tissue processing and microscopic evaluation was required, retained tissues will be discarded after finalisation of the main study report.

Postmortem examinations (offspring):

GROSS NECROPSY

Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities (especially of reproductive organs). After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs.

BLOOD SAMPLING for Thyroid Hormone Analysis:

For potential thyroid hormone analysis, blood samples were taken by decapitation into tubes containing K3-EDTA as anticoagulant as follows:
• from up to two pups per litter on PND4 (females if possible),

The collected pup blood (plasma) samples were pooled by litter.
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. Timing are documented in the raw data.
Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4°C). The resulting plasma was divided in at least two aliquots (volume target was at least 150 μL for the first aliquot and at least 150 µL for the second aliquot, any remaining sample (if any) was kept as a third aliquot) and stored in an ultra-freezer (-80±10°C) until analysis.
Thyroid hormone analysis was not required by the Sponsor’s Representative.

HISTOLOGY and MICROSCOPIC EVALUATION

No histopathology evaluation was performed.
If no tissue processing and microscopic evaluation was required, retained tissues will be discarded after finalisation of the main study report.

ANOGENITAL DISTANCE, NIPPLE RETENTION

No test item related effect was observed on anogenital distance or nipple retention during the study

No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control

There was no nipples/areolae presence seen in any of the male pups on PND 13

Statistics:

Parental Males
- Body weight and body weight gain
- Food consumption
- Number of fertile pairings
- Number of infertile males
- Male mating index
- Male fertility index
- Organ weights (absolute and relative to the body and brain weights)

Parental Females
- Body weight and body weight gain
- Food consumption
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non-mated females
- Female mating index
- Female fertility index
- Gestation index
- Duration of pregnancy (days)
- Number of corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups on Day 0, 4 and 13
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)

Offspring
- Mean pup body weight (per pup within the group and per litter) on PND 0, 4 and 13
- Mean pup body weight gain (per litter) between PND 0-4, PND 4-13 and PND 0-13
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0, 4 and 13
- Survival Index of pups on postnatal Days 0, 4 and 13
- Sex ratio % on postnatal Days 0, 4 and 13
- Anogenital distance, nipple retention

Reproductive indices:

Reproductive Ability Assessment and Indices:

There were test item related adverse effects for the Ultra High dose group with regard to reproductive ability, mating or gestation indices.

The mating index was 100% in all groups (males and females). The fertility index was 100%, 83%, 91%, 92% and 67% in the Control, Low, Mid, High and Ultra High dose groups for males and 100%, 83%, 91%, 92% and 25% in the Control, Low, Mid, High and Ultra High dose females. The gestation index was 100% in all groups (all pregnant animals produced litters).

Eight females in the Ultra High dose group were non pregnant. It was considered as test item related effect.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 6 days of pairing (cohabitation). The mean duration of mating was 2.58, 2.83, 3.00, 3.08 and 4.17 days in the Control, Low, Mid, High and Ultra High dose groups, respectively.

Offspring viability indices:

Index Calculation


Survival Index %
( Number of live pups (at designated time) / Number of pups born) x100

Survival index on PND13 will be calculated from number of pups after culling on PND4 instead of number of pups born.

Pre-implantation mortality %

((Number of Corpora lutea - Number of implantations)/ Number of corpora lutea) x100)

Intrauterine mortality %

((Number of implantations - number of liveborns)/Number of implantations) x100

Total mortality %
((("Number of implantations-Number of viable pups (PND0/4/13)) /Number of implantations) x 100

Sex ratio % (females)

((Number of female pups (PND0/4/13)) /Number of viable pups (PND0/4/13)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased activity was observed for 1/12 Ultra High dose females from Day 37 until Day 45 (9 days).
Hunched back was observed for 2/12 Ultra High dose females from Day 11 until Day 48. The maximum longevity of the symptom was 31 days.
Noisy respiration was observed for 1/12 High dose male on Day 9 and from Day 25 to Day 26.
Piloerection was observed in case of 4/12 High dose females from Day 34 until Day 48, for 4/12 High dose males from Day 3 to Day 4, for 4/12 Ultra High dose males on Day 3, Day 4, and from Day 11 to Day 12 and 12/12 Ultra High dose females from Day 3 until Day 48. The maximum longevity of the symptom was 37 days.

Decreased activity, hunched back, noisy respiration and piloerection were considered as test item related effects, the following findings were all considered as background findings.

Red discharge (left eye) was observed for 1/12 Control male from Day 10 to Day 20.
Scar was observed for 1/12 Control from Day 2 to Day 6, for 1/12 Mid dose male from Day 2 to Day 6, for 1/12 High dose male from Day 2 to Day 16, for 1/12 Mid dose female from Day 14 to Day 16 and for 1/12 Ultra High dose females from Day 15 until Day 29.
Wound was observed for 1/12 High dose male from Day 2 to Day 4 and 1/12 Ultra High dose female on Day 14.
Increased salivation was observed for 2/12 High dose female from Day 32 until Day 48 and for 1/12 Ultra High dose female from Day 44 until Day 48.
Alopecia (neck dorsa area) was observed for 1/12 Control female from Day 30 until euthanasia.
Thin fur (right cheek) was observed 1/12 Mid dose female from Day 14 to Day 16.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Mid dose female was found dead on Day 14 and one Ultra High dose female was found dead on Day 19. The animal had no clinical signs prior death, the animal had piloerection on Day 3 Based on the necropsy findings, gavage accident was considered as a possible cause of death for both animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly decreased body weight was observed during the first week of treatment in the High and Ultra High dose male groups (p<0.05) but the animals had the approximately same growth rate as controls thereafter. The body weight effect was test item related but transient.
Statistically significantly decreased body weight gain was observed in the High (p<0.05) and Ultra High (p<0.01) dose males during the first week.
In females, there was no effect on body weight or gain in the first 14 days of the study. Statistically significantly decreased body weight gain was observed in the High (-26.2%, p<0.01) dose female group during gestation.
Decreased body weight gain (-24.7%) was observed for the Ultra High dose females during gestation without statistical significance (n=3 due to the low pregnancy rate).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in the food consumption was recorded for the High (p<0.05 and p<0.01) and Ultra High dose (p<0.01) males, Ultra High dose females (p<0.01) from Day 0 to Day 14 and during gestation for High dose females (p<0.01).
Based on the body weight gain and food consumption data, the observed effects for the High and Ultra High dose females during gestation were considered as test item related adverse effect.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Decreased activity was observed for 1/12 Ultra High dose females from Day 37 until Day 45 (9 days).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 3.92-4.15 days was observed in the different groups) before starting the treatment period.
Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data during the pre-mating and mating periods (mean cycle length was 4.00, 4.03, 4.08, 4.13 and 4.23 days in the Control, Low, Mid, High and Ultra-High dose groups, respectively).
Prolonged oestrus was recorded for one (1/12) High and three (3/12) Ultra-High dose females. The incidence in the Ultra High dose was considered as being a test item related effect.
Prolonged dioestrus was noted for one Low dose female (#2505), one Mid dose female and four Ultra High dose females. Statistically significantly increase of the number of Ultra High dose females in prolonged dioestrus was considered as being a test item related effect as it is outside the historical range.
Pseudo-pregnancy was noted for 1, 2, 3 and 2 in Low, Mid, High and Ultra-High dose females but this frequency was in line with the normal, expected range.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were test item related adverse effects for the Ultra High dose group with regard to reproductive ability, mating or gestation indices.
The mating index was 100% in all groups (males and females). The fertility index was 100%, 83%, 91%, 92% and 67% in the Control, Low, Mid, High and Ultra High dose groups for males and 100%, 83%, 91%, 92% and 25% in the Control, Low, Mid, High and Ultra High dose females. The gestation index was 100% in all groups (all pregnant animals produced litters).
Eight females in the Ultra High dose group were non pregnant. It was considered as test item related effect.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 6 days of pairing (cohabitation). The mean duration of mating was 2.58, 2.83, 3.00, 3.08 and 4.17 days in the Control, Low, Mid, High and Ultra High dose groups, respectively.

Details on results (P0)

Evaluation of the Gestation, Parturition and Post-partum Period
The mean duration of pregnancy was comparable in the Control and test item treated groups (actual values are listed in Table Summary of the pregnancy evaluation).
As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.
The number of implantation sites was statistically significantly decreased in the High (p<0.05) and in the Ultra High dose (p<0.01) groups compared to the control mean. The data were outside of the historical control range (mean ± SD = 16.1 ± 5.2) in case of the Ultra High dose group, hence it was considered as test item related clearly adverse effect; the similar but lesser effect in the High dose group appears to be treatment related.
There was statistically significant decrease on the number of pups born in the High (p<0.01) and in the Ultra High (p<0.05) dose groups, the value for the Ultra High dose group was at the lower limit of the normal range, hence it is considered as test item related adverse effect (for more details see Table Summary of the pregnancy evaluation).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive effects

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Based on effects on fertility, implantation and pups born

2

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other:

Remarks on result:

other: based on relatively minor effects in clinical symptoms, body weight gain and food consumption

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a test item related effect on mortality or survival of the pups (F1 generation).
The mean number of viable pups were statistically significantly decreased in the High and Ultra High dose groups (p<0.01 and p<0.05), data were outside of the historical control range for the High and Ultra High dose groups, and it is considered as test item related effect.
Pre-natal mortality, mean for the High dose group was statistically significantly changed (p<0.05) compared to the Control.
There were no other significant differences or effects that could be ascribed to treatment on the post-natal mortality values in any of the dose groups.
Evidence of suckling was recorded for all live born pups in the study.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a test item related effect on mortality or survival of the pups (F1 generation).
The mean number of viable pups were statistically significantly decreased in the High and Ultra High dose groups (p<0.01 and p<0.05), data were outside of the historical control range for the High and Ultra High dose groups, and it is considered as test item related effect.
Pre-natal mortality, mean for the High dose group was statistically significantly changed (p<0.05) compared to the Control.
There were no other significant differences or effects that could be ascribed to treatment on the post-natal mortality values in any of the dose groups.
Evidence of suckling was recorded for all live born pups in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences in the offspring body weights or weight gains.
Statistically significantly increased litter mean male pup body weight was observed on PND 0 and PND4 in case of the High dose group but without dose response. The data were within the historical control range.
There were statistically significantly decreased mean pup body weight gain per litter (PND4-PND13) in the Ultra High dose group (p<0.05 and p<0.01).

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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item related effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples/areolae presence seen in any of the male pups on PND 13

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related effects were observed in the organ weights of the test item treated female and male animals compared to controls.
Terminal body weights of animals were not statistically significantly different between the groups. There were no treatment-related statistically significant differences among groups of females in the weights of organs measured when compared to Controls.
Weights of sex organs (absolute, body and brain related) in the Ultra High dose male group were all lower than the Controls, but without statistical significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

TERMINAL EUTHANASIA / F1 Generation (PND 13)
Macroscopic Findings
No test item-related macroscopic findings were observed.

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

Remarks:

pup development and survival

Generation:

F1

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

other:

Remarks on result:

other: normal post-natal mortality and reduced pup growth

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

PARENTAL

 

 

Selected body weight parameters of parental animals

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Number of animals	12	12	12	12	12
Males, Body weight on Day 14 (g)	476.9	476.4	475.5	448.8*	448.1*	DN
	difference (%)	-0.1	-0.3	-5.9	-6.0
Males, Body weight on Day 27 (g)	513.8	518.0	511.4	486.1	483.5	NS
	difference (%)	0.8	-0.5	-5.4	-5.9
Males, Body weight gain Day 0-7 (g)	10.3	8.6	6.3	-6.4*	-9.4**	DN
	difference (%)	-16.9	-38.7	-162.1	-191.1
Males, Body weight gain Day 7-27 (g)	57.8	64.3	60.1	48.9	48.1	NS
	difference (%)	11.1	3.9	-15.4	-16.9
Males, Body weight gain Day 0-27 (g)	68.2	72.8	66.4	42.5*	38.7*	DN
	difference (%)	6.8	-2.6	-37.7	-43.3
Number of animals	12	10	10	11	3
Females, Body weight on GD 20 (g)	458.3	446.6	454.7	415.9	405.3	NS
	difference (%)	-2.5	-0.8	-9.2	-11.5
Females, Body weight gain GD 0-20 (g)	179.7	161.6	169.7	132.5**	135.3	DU
	difference (%)	-10.1	-5.5	-26.2	-24.7
Females, Body weight on PPD 13 (g)	387.8	380.0	383.3	382.7	371.0	NS
	difference (%)	-2.0	-1.2	-1.3	-4.3
Females, Body weight gain PPD 0-13 (g)	36.4	37.2	41.8	50.5	42.7	NS
	difference (%)	2.2	14.8	38.7	17.2
Females, Body weight gain Day 0-PPD13 (g)	127.4	122.7	123.7	121.0	130.3	NS
	difference (%)	-3.7	-2.9	-5.0	2.0

GD: Gestation Day, PPD: Post-partum Day

DN: Dunnett’s Multiple Range Test; DU: Dunn test,

NS: Statistically not significant when compared to the control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01.

 

Selected daily food consumption parameters of parental animals (g/animal/day)

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Males, Food consumption Day 0-7 (g)	21.54	20.81	20.48	17.01*	15.40**	DU
	difference (%)	-3.4	-4.9	-21.0	-28.5
Males, Food consumption Day 7-14 (g)	23.57	23.68	23.65	20.52	19.46**	DN
	difference (%)	0.5	0.4	-12.9	-17.4
Males, Food consumption Day 14-27 (g)	24.86	24.91	24.38	21.91**	21.83**	DN
	difference (%)	0.2	-1.9	-11.9	-12.2
Females, Food consumption Day 0-14 (g)	15.75	15.64	16.60	15.82	12.55**	DN
	difference (%)	-0.7	5.4	0.5	-20.3
Females, Food consumption GD 0-20 (g)	24.84	24.04	23.72	21.46**	22.93	DN
	difference (%)	-3.2	-4.5	-13.6	-7.7
Females, Food consumption PPD 0-13 (g)	51.07	48.05	50.24	45.75	43.77	NS
	difference (%)	-5.9	-1.6	-10.4	-14.3

GD: Gestation Day, PPD: Post-partum Day

DN: Dunnett’s Multiple Range Test; DU: Dunn test,

NS: Statistically not significant when compared to the control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

 

Summary of reproductive parameters (males)

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Number of treated animals	12	12	11	12	12
Number of males used for mating	12	12	11	12	12
Number of pre-terminal death	0	0	1	0	0
Number of successful mating	12	12	11	12	12
Number of infertile animals	0	2	1	1	4*
Male mating index (%)	100	100	100	100	100
Male fertility index (%)	100	83	91	92	33

* Note: Although the males did not produce any implantations after mating, which classes the males as infertile, we can not exclude the very low female fertility of this group being the cause

 

Summary of reproductive parameters (females)

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Number of treated animals	12	12	12	12	12
Number of females used for mating	12	12	11	12	12
Number of sperm positive females	12	12	11	12	12
Number of females with no implantation sites	0	2	1	1	8
Number of females with no corpora lutea	0	2	1	1	4
Number of pregnant females	12	10	10	11	3
Number of found dead pregnant	0	0	0	0	1
Number of pregnant females with live born(s)	12	10	10	10	3
Number of pregnant females with no pups	0	0	0	1	0
Female mating index (%)	100	100	100	100	100
Female fertility index (%)	100	83	91	92	25
Female gestation index (%)	100	100	100	100	100

Notes: Two non-pregnant females were observed in the Low dose group, one in the Mid dose group, one in the High dose group and eight in the Ultra-High dose group had total intrauterine mortality, but because corpora lutea were present they were counted as fertile.

 

Summary of the pregnancy evaluation

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Number of evaluated females	12	10	10	10	3
Number of pregnant females	12	10	10	11	3
Duration of pregnancy (days)	22.25	22.30	22.20	22.60	22.67	NS
Number of implantations, mean	17.75	13.75	16.55	12.17*	3.45**	U
Number of pups born, mean	16.50	14.80	16.90	12.10**	10.00*	D
Number of live born pups, mean	16.50	14.70	16.80	10.91**	10.00*	U
Pre-natal mortality, mean	1.25	1.80	1.40	2.36	2.67	NS
Pre-natal mortality (PND0-13) (%), mean	6.92	11.47	7.68	25.20*	19.42	U
Post-natal mortality (PND0-13), mean	0.50	0.40	0.40	0.30	0.00	NS
Post-natal mortality (%), mean	2.91	2.35	2.05	1.61	0.00	NS
Total mortality (PND0-13), mean	1.75	2.20	1.80	2.60	2.67	NS
Total mortality (PND0-13) (%), mean	9.60	13.56	9.59	19.25	19.42	NS

D: Duncan’s Multiple Range Test; U: Mann-Whitney U-test,

NS: Statistically not significant when compared to the control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01.

 

OFFSPRING

Summary of the offspring mortality

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Number of evaluated litters	12	10	10	10	3
Number of pups born	198/12	148/10	169/10	121/10**	30/3**	CH
Number of cannibalized pups	1/1	2/1	2/1	1/1	0/0	NA
Number of autolyzed pups	5/3	3/3	3/2	3/3	0/0	NA
Number of stillborn pups	0/0	0/0	0/0	0/0	0/0	NA
Number of live born pups	198/12	147/10	168/10	120/10	30/3	NA
Number of found dead pups (born alive)	0/0	0/0	0/0	0/0	0/0	NA
Number of living pups on PND0	198/12	147/10	168/10	120/10	30/3	NA
Number of cannibalized pups (PND0-13)	1/1	2/1	2/1	1/1	0/0	NA
Number of found dead, intact pups (PND0-13)	0/0	0/0	0/0	0/0	0/0	NA
Total number of pups died (born alive)	6	4	4	3	0	NS
Culled for blood sampling on PND4	24/12	18/9	19/10	16/8	2/1	NA
Culled for litter standardization on PND4	48/12	31/8	45/10	13/4	1/1	NA
Number of viable pups on PND13	120/12	94/10	100/10	88/10*	27/3**	CH

PND: Post-natal day, NS: Statistically not significant when compared to the control, NA: Not applicable

Statistical significance compared to control: * = p<0.05, ** = p<0.01, CH: Chi Square

 

Summary of survival (offspring)

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Number of evaluated litters	12	10	10	10	3
Number of pups born, mean	16.50	14.80	16.90	12.10**	10.00*	D
Number of live born pups, mean	16.50	14.70	16.80	10.91**	10.00*	U
Number of living pups on PND13, mean	10.00	9.40	10.00	8.80	9.00	NS
Pre-natal mortality, mean	1.25	1.80	1.40	2.36	2.67	NS
Pre-natal mortality (%), mean	6.92	11.47	7.68	25.20*	19.42	U
Post-natal mortality on PND0-4, mean	0.50	0.40	0.40	0.20	0.00	NS
Post-natal mortality on PND0-4 (%), mean	2.91	2.35	2.05	1.08	0.00	NS
Total mortality on PND4, mean	1.75	2.20	1.80	2.50	2.67	NS
Total mortality on PND4 (%), mean	9.60	13.56	9.59	18.75	19.42	NS
Post-natal mortality on PND0-13, mean	0.50	0.40	0.40	0.30	0.00	NS
Post-natal mortality on PND0-13 (%), mean	2.91	2.35	2.05	1.61	0.00	NS
Total mortality on PND13, mean	1.75	2.20	1.80	2.60	2.67	NS
Total mortality on PND13 (%), mean	9.60	13.56	9.59	19.25	19.42	NS
Survival index on PND0	100.00	99.41	99.50	99.23	100.00	NS
Sex ratio % (Female) on PND0	51.44	46.75	54.52	48.67	47.33	NS
Survival index on PND4	97.09	97.65	97.95	98.92	100.00	NS
Sex ratio % (Female) on PND4	52.01	46.83	54.48	48.51	47.33	NS
Survival index on PND13	100.00	100.00	100.00	99.00	100.00	NS
Sex ratio % (Female) on PND13	50.00	48.50	53.00	51.94	48.61	NS

D: Duncan’s Multiple Range Test; U: Mann-Whitney U-test Versus Control,

NS: Statistically not significant when compared to the control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01.

 

Anogenital distance

 

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Male pups
Number of evaluated litters	12	10	10	10	3
Anogenital distance, litter mean of males (mm)	3.60	3.58	3.62	3.88	3.92	NS
Minimum / Maximum value, litter mean (mm)	3.2/3.8	3.3/3.9	3.1/4.2	3.4/4.2	3.7/4.1
Female pups
Number of evaluated litters	12	10	10	10	3
Anogenital distance, litter mean of females (mm)	1.77	1.82	1.75	1.87	1.85	NS
Minimum / Maximum value, litter mean (mm)	1.6/1.9	1.7/2.3	1.5/1.9	1.6/2.3	1.7/2.0

NS: Statistically not significant when compared to the control

 

Selected body weight data (offspring)

 

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (12.5)	Mid dose (50)	High dose (200)	Ultra-High dose (800)
Number of evaluated litters	12	10	10	10	3
Mean litter body weight (PND0), g	6.662	6.972	6.739	7.483	7.453	NS
Mean litter body weight (PND4), g	9.927	10.346	9.736	11.553	11.780	NS
Mean litter body weight gain (PND0-4), g	3.264	3.353	2.987	4.057	4.327	NS
Mean litter body weight (PND13), g	29.409	30.744	29.125	29.789	27.160	NS
Mean litter body weight gain (PND4-13), g	19.466	20.340	19.269	18.136	15.346**	DN
Mean litter body weight gain (PND0-13), g	22.725	23.698	22.330	22.246	19.674	NS

PND: Post-natal day, DN: Dunnett’s Multiple Range Test; DU: Dunn test, NS: Statistically not significant when compared to the control. Statistical significance compared to control: * = p<0.05, ** = p<0.01

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic toxicity of the parental generation was considered to be
200 mg/kg bw/day
(based on relatively minor differences in clinical symptoms, body weight gain and food consumption).
The NOAEL for reproductive effects of the parental generation was considered to be 50 mg/kg bw/day
(based on effects on fertility, implantation and pups born).
The NOAEL for pup development and survival was considered to be 200 mg/kg bw/day
(based on effects on normal post-natal mortality but reduced pup growth).

Executive summary:

The purpose of this OECD No. 421 study was to obtain information on the possible toxic effects of BAB (Benzene, mono-C11-C13-branched alkyl derivatives) test item following repeated (daily) administration by oral gavage to Sprague Dawley (SD) rats at 4 dose levels. A control group received the vehicle only (Propylene glycol + 1% Polysorbate).

Male and female SD rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general and/or weekly detailed observation of clinical signs, weekly body weight and food consumption. The reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal day (PND13). At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals and/or offspring.

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of an OECD 407 study (CIT/Study No. 36997 TSR, [8]) and 90 days toxicity test (FR – 11995.307.001.13[9]). Based on these data, 800 mg/kg bw/day was chosen as High dose level in this study. Lower doses were spaced with a factor of 4.

 

Experimental design

Group Number	Group designation	Dose level (mg/kg bw/day)	Dose formulation concentration (mg/mL)	Dose formulation volume (mL/kg bw)	Animal numbers
					Male	Female
1	Control	-	-	5	1001-1012	1501-1512
2	Low Dose	12.5	2.5		2001-2012	2501-2512
3	Mid Dose	50	10		3001-3012	3501-3512
4	High Dose	200	40		4001-4012	4501-4512
5	Ultra-High Dose	800	160		5001-5012	5501-5512

 

In summary, under the conditions of this study the daily administration of BAB (Benzene, mono-C11-C13-branched alkyl derivatives) by oral gavage to SD rats at dose levels of 12.5, 50, 200 and 800 mg/kg bw/day (Low, Mid, High and Ultra High dose groups, respectively) did not result in test item related mortality for adult animals.

Test item related clinical symptoms (decreased activity, hunched back, noisy respiration and piloerection in 1 to 4/12 animals per group) were observed mainly in the High and Ultra High dose parental groups.

There was a body weight loss in High & Ultra High males in the first 7 days, with a low food intake but based on the body weight gain and food consumption data was close to controls thereafter.

The High and Ultra High dose females were unaffected up until mating, but had reduced growth during gestation, which was probably related to the lower litter sizes in these animals.

The increased incidence of Ultra High dose females in prolonged dioestrus and prolonged oestrus was considered as being a test item related effect (probably related to the low pregnancy rate).
There were test item related adverse effects for the Ultra High dose group with regard to fertility index, with only 3/12 females producing a litter.
The number of females in the Ultra High dose group with no corpora lutea was relatively high, mean numbers of implantation sites was statistically significantly decreased and well below the historic control data.
The High dose females were partially affected with lower implantation numbers. These were considered as test item related adverse effects; in the absence of any signs of maternal toxicity at the time of mating these appear to be a direct effect on the reproduction system.

There were no adverse effects on the F1 offspring clinical signs, physical or sexual development. There were very low number of litters in the Ultra High dose group. The Low and Mid dose groups were unaffected. There were test item related adverse effects on the mean number of pups born and the number of viable pups in the Ultra High dose group; the High dose group was affected to a lesser extent.

No test item related macroscopic finding was recorded for F1 pups at necropsy.

No test item related macroscopic changes were recorded at necropsy of routine organs/tissues or in any reproductive organs.

Under the experimental conditions of this study and based on the results thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis and reproductive performance, there was no evidence for any endocrine effects.

 

The NOAEL for systemic toxicity of the parental generation was considered to be
200 mg/kg bw/day
(based on relatively minor differences in clinical symptoms, body weight gain and food consumption).

The NOAEL for reproductive effects of the parental generation was considered to be 50 mg/kg bw/day
(based on effects on fertility, implantation and pups born).

The NOAEL for pup development and survival was considered to be 200 mg/kg bw/day
(based on normal post-natal mortality but reduced pup growth).