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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 nov 2013 to 09 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD 422 under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Fatty acids, tall oil and rosin, reaction products with maleic anhydride
IUPAC Name:
Fatty acids, tall oil and rosin, reaction products with maleic anhydride
Constituent 2
Chemical structure
Reference substance name:
Fatty acids, tall oil and rosin reacted with maleic anhydride
EC Number:
940-281-5
Molecular formula:
Generally varies from C18H34O2 to C24H32O5
IUPAC Name:
Fatty acids, tall oil and rosin reacted with maleic anhydride
Test material form:
other: viscous liquid
Details on test material:
Identification: Fennoanz M30
Storage Conditions: Dry, at room temperature (20 ± 5 ºC) and protected from light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Models, S.L. (Spain)
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Males: 271-321 g; Females: 198-221 g
- Fasting period before study: none
- Housing: Pretest and premating period: maximum 5 animals/cage; Mating period: one male and one female/cage; Postmating: individually
- Diet: Pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet ad libitum
- Water: Tap water in bottles ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ºC
- Humidity (%): 40-65%
- Air changes (per hr): 15-20/hr
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily or every 7 days kept under strirring

VEHICLE: corn oil
- Justification for use and choice of vehicle (if other than water): not indicated
- Concentration in vehicle: 18.75, 62.50 and 187.50 mg/mL
- Amount of vehicle (if gavage): 4 mL
- Lot/batch no. :MKBH4894V
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 18 hours
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
System suitability (CV 0.9-1.4 %), linearity (R2 0.998), accuracy (92.4-96.5%) and repeatability (CV 0.6-2.1%) and specifity (no interference with the vehicle) were found to be accurate
System:
HPLC System: HPLC separation module Alliance 2695 (Waters)
UV Detector: HPLC dual wavelength ultraviolet detector 2487 (Waters)
Column: Symmetry® C 5µm (3.9 x 150 mm) (Waters)
Injection volume: 25 µL
Mobile Phase: Eluent A: 0.05% Phosphoric acid in Water; Eluent B: Acetonitrile
Column flow rate: 1.2 mL/min
Retention time: Approx. 16.3 min
Detector wavelength: 205 nm

Calibration solution: 2 g of the test substance (Density: 1.04) was weighed in a 60-mL polypropylene tube. 18.08 mL (20-1.92 mL) of corn oil was added to give a final concentration of 100 mg /mL. The solution was left stirring for at least 30 minutes (using a propeller stirrer) and was protected from light.

Analysis for accuracy of preparation, homogenicity and stability:
accuracy of preparation on first week of treatment (28 November 2013: 90.2-96.6 % of nominal (75 and 750 mg/kg bw)
accuracy of preparationpost-pairing period (7 January 2014): 87.7-94.7% of nominal (75, 250 and 750 mg/kg bw)
homogenicity: 94.1 ± 2.3% (at 75 mg/kg bw) and 92.8 ± 2.9% (at 750 mg/kg bw)
Stability (28 November 2013): mean deviation from T0 5.2-19% over 6 days
Duration of treatment / exposure:
Males: two weeks prior to mating and at least up to and including the day before sacrifice (at least a total of 42 days).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 post partum).
Frequency of treatment:
daily
Details on study schedule:
Parental animals not mated until day 18
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 250 and 750 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
12/sex
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on a 14-day dose-range-finding toxicity study in rats (Harlan Laboratories Study S46418 14-Day Oral (gavage) Dose-Range-Finding Toxicity Study in the Wistar Rat), using dose levels of 0, 100, 500 and 1000 mg/kg/day.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes (Defecation, Urination, Muscular tone, Appareance, Skin, Respiration, Ruffled fur, Lachrymation, Salivation, Grasp response, Gait, Hunched back )
- Time schedule: once weekly in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations:
P males: at pretest, twice weekly during the pre-pairing and pairing period and daily during the postpairing period.
P females: at pretest, twice weekly during the pre-pairing and pairing period and daily until day 4-5 post partum.

FOOD CONSUMPTION : yes
- Time schedule for examinations:
P males: at pretest, once weekly during the pre-pairing period and two weeks during the postpairing period.
P females: at pretest, once weekly during the pre-pairing period and days 0-7, 7-14, 14-21 post coitum and days 1-4 post partum.

WATER CONSUMPTION : no

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
5 selected males: on day 43 of treatment (on day of sacrifice)
5 selected females: on day 5 post partum (on day of sacrifice)
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: No
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Hemoglobin concentration distribution width, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Total leukocyte count, Differential leukocyte count (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Large Unstained Cells)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
5 selected males: on day 43 of treatment (on day of sacrifice)
5 selected females: on day 5 post partum (on day of sacrifice)
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: No
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin(total), Cholesterol(total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatine kinase, Gamma-glutamyl-transferase, Calcium, Inorganic Phosphorus, Sodium, Potassium, Chloride, Protein(total), Albumin, Globulin, Albumin/Globulin ratio, Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: 5 males/dose last week of treatment, 5 females/dose day 4 post-partum
- Battery of functions tested: motor activity, grip strength, palpebral , iridial reflex, push-off (hind legs), pain response, Startle/hearing, righting reflex
Oestrous cyclicity (parental animals):
vaginal smears were collected during the mating period
Sperm parameters (parental animals):
spermatology stage evaluation of control and high dose males: testes and epididymides
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups live births, postnatal mortality, presence of gross anomalies, rightening reflex

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes : on all animals
external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents

HISTOPATHOLOGY: Yes : on 5 animals/sex of the control and high dose group
Adrenal glands, Aorta, Bone marrow (femur) (air-dried), Brain (bulbar, cerebellar and cortical sections), Epididymides (Bouin's solution), Esophagus, Eyes with optic nerve (Davidson’s fixative), Femur (with articular surface), Heart (with papillary muscle), Intestine, large (cecum, colon and rectum), Intestine, small (duodenum, jejunum and ileum), Kidneys, Larynx, Liver, Lungs, with main bronchi and bronchioles, Lymph nodes (mandibular and mesenteric), Mammary gland area, Nose (the entire head will be collected), Ovaries, Pancreas, Peyer’s patches, Pituitary gland, Prostate, coagulating gland and seminal vesicles, Salivary glands (mandibular, sublingual and parotid), Sciatic nerve, Skeletal muscle, Skin (abdominal), Spinal cord (cervical, thoracic and lumbar), Spleen, Sternum (with bone marrow), Stomach (glandular and nonglandular), Testes (Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder, Uterus (cervix, corpus and oviducts), Vagina, All gross lesions
Other animals gross lesions (if any)
Organ weights: on all animals (unless found dead or killed in extremis): Adrenals, Kidneys, Pituitary, Testes, Brain, Liver, Prostate and seminal vesicle, Thymus, Epididymides, Lungs, Salivary glands (mandibular), Thyroid and parathyroids, Heart, Ovaries, Spleen, Uterus and oviducts
Postmortem examinations (offspring):
GROSS NECROPSY
Statistics:
•The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied when the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex (Dunnett, 1955).
•The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution (Miller, 1981).
•Fisher's exact-test was applied to the macroscopic findings (Gad, 1986).
•Armitage/Cochran Trend Test for non-neoplastic lesions, if appropriate (Armitage, 1955).
•Bonferroni-test was applied to some reproduction parameters.
Reproductive indices:
Percentage Mating (%) = (Number of females mated/Number of females paired) x 100
Conception rate (%) = (Number of females achieving pregnancy/Number of females mated) x 100
Fertility index (%) = (Number of females achieving pregnancy/Number of females paired) x 100
Gestation Index (%) = (Number of females with live pups/Number of females pregnant) x 100



Offspring viability indices:
Viability index (%) = (Number of alive pups on Day 4/Number of offspring alive on Day 1)x 100
Live birth index (%) = (Number of offspring alive on Day 1/ Number of offspring delivered)x 100




Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
at 750 mg/kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 750 mg/kg bw
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at 750 mg/kg bw
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at 250 and 750 mg/kg bw

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY:
Males: 750 mg/kg bw: 1
Females: 250 mg/kg bw : (terminated post partum 1)
750 mg/kg bw: 3 at parturation; (terminated post-coital 1; post partum 4)
Salivation in males and females at 250 mg/kg bw and 750 mg/kg bw; rufled fur in males at 750 mg/kg bw from week 3 onwards and in females at 250 mg/kg bw and 750 mg/kg bw from week 4 onwards

BODY WEIGHT AND WEIGHT GAIN:
Males: 75 mg/kg bw significantly decreased
250 mg/kg bw decreased
750 mg/kg bw significantly decreased
No clear dose response and within normal ranges
Females: 750 mg/kg bw: significantly decreased

FOOD CONSUMPTION :
Males: 750 mg/kg bw significantly decreased (within normal ranges)
Females: 750 mg/kg bw significantly decreased

HAEMATOLOGY:
Males: 750 mg/kg bw: Hb significantly decreased: HCT decreased; Reticulocytes significantly increased; Platelets decreased; APTT significantly increased; MCHC/MCH (significantly) decreased; HDW significantly decreased: RDW significantly increased
Females: 250 and 750 mg/kg bw: MCHC/MCH significantly decreased
Effects in males were within background ranges

CLINICAL CHEMISTRY:
No treatment related effects

NEUROBEHAVIOUR:
Males: 250 mg/kg bw decreased motor activity; significantly increased hind limb grip strength
Females: 250 mg/kg bw significantly increased hind limb grip strength
No dose response relationship

Sensory reactivity assessments: no treatment related effects

ORGAN WEIGHTS:
Males: 750 mg/kg bw: liver significantly increased; kidney significantly increased (rel to BW); thymus significantly decreased
Females: 750 mg/kg bw: liver significantly increased; kidney significantly increased (rel to BW); thymus significantly decreased; adrenals significantly
increased
250 mg/kg bw: liver significantly increased (rel to BW)

GROSS PATHOLOGY:
Males: control: testes small (1)
75 mg/kg bw: thymus foci (3)
250 mg/kg bw: thymus foci (3)
750 mg/kg bw: thickened stomach wall with foci (6/4), discoloured caecum (7), discoloured mesenteric lymphnodes (6)
Females: control: thymus small (4)
75 mg/kg bw: thymus small (6), stomach discoloured (5)
250 mg/kg bw: thymus small (6), stomach discoloured (3)
750 mg/kg bw: thickened stomach wall with foci (4/2), dilated duodecum (4), small thymus(9)

HISTOPATHOLOGY:
Males: control: thymus atrophy (5), testis atrophy (1), epididymides aspermia (1)
75 mg/kg bw: thymus atrophy (3)
250 mg/kg bw: stomach inflammation(2); thymus atrophy (3)
750 mg/kg bw: stomach inflammation/ulceration/acanthosis (6/1/10); thymus atrophy (6) ;bladder mono-
nuclear foci/hyperplasia (2/3): spleen lymphoid depletion (1)
Females: control: thymus atrophy (6)
75 mg/kg bw: thymus atrophy (6); stomach inflammation/ulceration (1/1)
250 mg/kg bw: thymus atrophy (6); stomach inflammation/ulceration/acanthosis (4/1/2); spleen lymphoid depletion (1)
750 mg/kg bw: thymus atrophy (10); stomach inflammation/ulceration/acanthosis (8/8/4); spleen lymphoid depletion (4); bladder mono-
nuclear foci/hyperplasia (4/4): kidney tubular vacuolisation (2); adrenals hypertrophy (4)
The effects on the thymus and the stomach showed a relation with dose regarding the incidence and severity of the effects in the 250 and 750 mg/kg bw dose groups. The effect on the thymus (and lymphoid depletion) may be stress related.

SPERM PARAMETERS (at control and 750 mg/kg bw):
testes: within normal ranges
epididymides: within normal ranges

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality, clinical signs, lower food consumption and body weight
Dose descriptor:
NOEL
Effect level:
>= 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: difficulties at parturation and breeding behaviour
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mating index; fertility index; number of implantation sites; duration of pregnancy; pregnancy index; sex ratio; sperm characterization

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
at 750 mg/ kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 750 mg/kg bw
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY INDEX (OFFSPRING): 97.7%, 98,2%, 90.4%, 48.4% at 0, 75, 250 and 750 mg/kg bw

LIVE BIRTH INDEX (OFFSPRING): 97%, 100%, 86%, 43% at 0, 75, 250 and 750 mg/kg bw

POST-NATAL LOSS (OFFSPRING): 2.1%, 1.8%, 12.4%, 51.6% at 0, 75, 250 and 750 mg/kg bw (significant at 250 and 750 mg/kg bw)

BEHAVIOURAL EFFECTS (OFFSPRING): no treatment related effects in rightening reflex

BODY WEIGHT (OFFSPRING): day 1 6.0, 6.6, 5.7, 4.8 g at 0, 75, 250 and 750 mg/kg bw
day 4 8.3, 8.5, 7.9, 5.9 g at 0, 75, 250 and 750 mg/kg bw

SEX RATIO (OFFSPRING): no treatment related effects

MACROSCOPY (OFFSPRING): no treatment related effects

OTHER FINDINGS (OFFSPRING): milk in stomach in 99-100% of pups at 0, 75 and 250 mg/kg bw, in 55-100% at 750 mg/kg bw (significantly decreased on day 1 and 2)

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on motor development, no macroscopic findings, lowered body weights were related to the maternal effect on nursing behaviour

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Averages maternal animals

pregnancy: 9, 11, 11, 11 at 0, 75, 250 and 750 mg/kg bw

corpora lutea: 14.0, 14.8, 13.9, 13.5 at 0, 75, 250 and 750 mg/kg bw

implants: 12.3, 12.3, 11.3, 11.8 at 0, 75, 250 and 750 mg/kg bw

pre-coital time: increased at 250 mg/kg bw and 750 mg/kg bw (near normal ranges)

females with live offspring (day 1): 9, 10, 11, 5 at 0, 75, 250 and 750 mg/kg bw

females with live young (day 4): 9, 10, 10, 3 at 0, 75, 250 and 750 mg/kg bw

Percentage Mating: 100% at all dose levels

Conception rate: 75.0%, 83.3%, 91.7%, 91.7% at 0, 75, 250 and 750 mg/kg bw

Fertility index: 75.0%, 83.3%, 91.7%, 91.7% at 0, 75, 250 and 750 mg/kg bw

Gestation Index: 100%, 100%, 100%, 45.5% at 0, 75, 250 and 750 mg/kg bw

Applicant's summary and conclusion

Conclusions:
The NOAEL on reproductive and developmental effects is 750 mg/kg bw.
The NOEL for maternal toxicity is set at 75 mg/kg bw based on effects on parturation and breeding
Executive summary:

Rats (12/sex/dose) received the test substance by gavage at 0, 75, 250 and 750 mg/kg bw daily for at least 6 weeks (including two weeks prior to mating, through mating, pregnancy and early lactation for females). The test design followed was according to OECD 422.

Mortality was reported among females at 750 mg/kg bw (during parturation or terminated during lactation). This mortality could not be related to specific toxicity, but food consumption and body weight were decreased. Clinical signs included ruffled fur and increased salivation. No apparent differences with control animals were noted for haematology, clinical chemistry, neurobehavioural assays and sensory reactivity.

At 750 mg/kg bw increased kidney (relative to body weight) and liver weights were observed. Necropsy revealed effects on the mucosa of the stomach at 250 and 750 mg/kg bw (males + females), thymus atrophy (males + females) and spleen lymphoid depletion (females) at all dose levels. The effect on the stomach is considered to be a local effect (local irritation). The effects on the thymus and spleen are considered to be stress related.

No effects were reported related to mating, fertility and reproduction (sperm quality, number corpora lutea, number implantation sites, pre- and postimplantion losses or sex ratio). At 250 mg/kg and 750 mg/kg a dose related increase in the number of females that died during delivery or failed to nurse their offspring .This is reflected in a lower gestation index, but considered to be related to generic non-specific toxicity in maternal animals.

An increase in the percentage of dead pups at first litter check was observed at 250 and 750 mg/kg. Consequently, the live birth index was 50% lower than that recorded in the control group at 750 mg/kg.. An increase in postnatal losses was also observed at 250 and 750 mg/kg. Therefore the viability index was lower. Both effects could be related to the difficulties females encountered during parturation. Pup body weight at 750 mg/kg bw was significantly lower, but this could be related to maternal toxicity. No effects on motor development and no macroscopic abnormalities were reported in any of the dose groups.

Based on the above mentioned the NOAEL for systemic effects in parental animals is 250 mg/kg bw. The NOAEL for effects on reproduction and development is 750 mg/kg bw. Based on the effects on breeding behaviour a NOEL of 75 mg/kg bw is considered to represent a worst case. This value will be taken as the starting point for the risk assessment.

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