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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-03-2013 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to the guideline and under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: extremely viscous liquid
Details on test material:
Identification : Fatty acids, tall-oil, reaction products with fatty acids C16-18 and C18-unsatd, branched and linear, diethylenetriamine and methylhydrogen maleate
Description : Amber coloured extremely viscous liquid
Purity : 100% (UVCB)
Storage Conditions : Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Han™:RccHan™:WIST strain
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: ca. 12 weeks
- Weight at study initiation: males 313 - 387g; females 192 - 236g,
- Housing: pre-mating (males post mating) 4/cage in solid floor polypropylene cages; during mating 1M/1F in polypropylene grid floor cages; gestation/lactation: individually in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes
- Diet: Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15%
- Air changes (per hr): ≥ 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Males: 14 days prior to mating; upto 14 days during mating and 14 days post-mating
Females: 14 days prior to mating; upto 14 days during mating; average 21 days of gestation; 4 days of lactation
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / 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: NA
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses By HPLC-MS (one test-item related peak)

Analytical standard: standard solution of 0.01 mg/L in methanol
Linearity: proven over 0-0.01638 mg/L
recovery: 98-102% of nominal (fortified samples)
accuracy of preparation : 93-109% of nominal (4 weekly samples)
stability over 21 days:93-97% of initial
Duration of treatment / exposure:
Males: 14 days prior to mating; upto 14 days during mating and 14 days post-mating
Females: 14 days prior to mating; upto 14 days during mating; average 21 days of gestation; 4 days of lactation
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12/sex
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on outcome of dose range finding study where no signs of toxicity were observed at 1000 mg/kg bw
Positive control:
NA

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: week days : before dosing, soon after dosing, and one hour and five hours after dosing
weekend and holidays: before dosing, soon after dosing, and one hour after dosing
- Cage side observations checked were included.in tables in the report

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly on all animals individually
- Check for Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration. Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, for females after mating on day 0, 7, 14 and 20 of gestation and day 1 and 4 of lactation

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly (cage basis), for females after mating on day 0, 7, 14 and 20 of gestation and day 1 and 4 of lactation

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes based on the cage side measurements of food consumption

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: daily during pre-mating period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males on day 42, females on day 4 of lactation
- Anaesthetic used for blood collection: No, withdrawal from the lateral tail vein
- Animals fasted: No
- How many animals: 5/sex of control and 1000 mg/kg bw groups
- Parameters checked: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), platelet count (PLT), Reticulocyte count (Retic), PTT and APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males on day 42, females on day 4 of lactation
- Anaesthetic used for blood collection: No, withdrawal from the lateral tail vein
- Animals fasted: No
- How many animals: 5/sex of control and 1000 mg/kg bw groups
- Parameters checked. Urea, Calcium (Ca++),,Glucose, Inorganic phosphorus (P),Total protein (Tot.Prot.),Aspartate aminotransferase (ASAT),Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: 5/sex of control and 1000 mg/kg bw groups
- Battery of functions tested: motor activity, hind/forelimb grip strength, sensory reactivitys (Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach)
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, detailed qualitative examination of the testes (the tubular stages of the spermatogenic cycle) in order to identify
missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, live births, postnatal mortality, weight (litter) , physical or behavioural abnormalities (righting reflex)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):
Sacrifice: males on day 43, non-pregant females on day 25 after mating, females and offspring on day 5 post-partum by overdose of pentobarbitone

GROSS PATHOLOGY: Yes
full necropsy on all animals. For females examination of the uterus for implementation sites and corpora lutea (if necessary Salewski staining)

ORGAN WEIGHTS:Yes of 5/sex of the control and high dose group
Adrenals, Prostate*, Brain, Seminal vesicles*, Epididymides*, Spleen, Heart, Testes*, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries*, Uterus (weighed with Cervix)*, Pituitary (post fixation)*
* weighed from all remaining animals

HISTOPATHOLOGY: Yes of 5/sex of the control and high dose group
Adrenals, Ovaries*, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary*, Bone & bone marrow (sternum), Prostate*, Brain (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland*, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles*,Epididymides*, Skin (hind limb), Eyes, Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions*, Heart, Spleen, Ileum (including peyer’s patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes*, Lungs (with bronchi), Thymus, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary gland*, Uterus/Cervix*, Muscle (skeletal), Vagina*

The tissues shown with * from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
macroscopy
Statistics:
Data were analysed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to
determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the MannWhitney U test (non-parametric).
Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant, this analysis was followed by pair-wise comparisons using Dunnett’s test. Where
Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also be investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Reproductive indices:
i. Pre-coital Interval

ii. Fertility Indices
-Mating Index (%)
-Pregnancy Index (%)

iii Gestation Length

iv. Parturition Index (%)

v Implantation Losses (%)
Offspring viability indices:
Live Birth and Viability Indices
Sex Ratio (% males)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
increased in females at 1000 mg/kg bw during gestation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
increased in females at 1000 mg/kg bw during gestation
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: gavage study

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY: no treatment related effects

BODY WEIGHT AND WEIGHT GAIN: at 1000 mg/kg bw increased in females during gestation

FOOD CONSUMPTION: at 1000 mg/kg bw increased in females during gestation

FOOD EFFICIENCY: no treatment related effects

WATER CONSUMPTION: at 300 and 1000 mg/kg bw increased in females

HAEMATOLOGY: decreased haematocrit in males at all dose levels (no dose-response relationship and within historical data)

CLINICAL CHEMISTRY: decreased creatinine in males at all dose levels (no dose-response relationship and within historical data)

NEUROBEHAVIOUR: no treatment related effects

ORGAN WEIGHTS: decreased thyroid weight in males at all dose levels (no dose-response relationship and within historical data), Increased weights of pituitary and ovaries in females at 1000 mg/kg bw

GROSS PATHOLOGY: no treatment related effects

HISTOPATHOLOGY: no treatment related effects
Qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle.

No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects related to the test substance were found at any of the doses tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING): no treatment related effects (viability index 98-100%; life birth index 99-100%))

SEX RATIO: no treatment related effects (41.4-54.1% males, within normal ranges)

CLINICAL SIGNS (OFFSPRING): no treatment related effects

Righting Reflex: No treatment related effects (79-93%, within normal ranges in all treatment groups)

BODY WEIGHT (OFFSPRING): increased litter weight at 1000 mg/kg bw on day 1 (increased number of pups)

GROSS PATHOLOGY (OFFSPRING); no treatment related abnormalities

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on the offspring were noted

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Pregnancy rate: 11, 10, 9 and 11 at 0, 100, 300 and 1000 mg/kg bw

Precoital interval: slightly prolonged at 300 and 1000 mg/kg bw From 1 -2 to 2 -3 days)

Gestation length: no treatment related effects (22 -23.5 day)

Mating Index: no treatment related effects (100%)

Corpora lutea: increased at 1000 mg/ kg bw

Implantation sites: increased at 1000 mg/kg bw

Pre-implantation loss: no treatment related effects

Post implantation loss: no treatment related effects

Number of offspring: increased at 1000 mg/kg bw

Applicant's summary and conclusion

Conclusions:
In a study according to OECD 422 no adverse effects were reported in parental animals and their offspring. No effects on reproductive performance were noted. The NOAEL for reproductive effects is 1000 mg/kg bw.
Executive summary:

Twelve rats/sex were treated with the test substance (oral gavage) at 0, 100, 300 and 1000 mg/kg bw during 14 days (pre-mating), during mating and 14 days thereafter (males). Females were further treated during gestation and until 5 days post partum. No adverse effects on mortality, clinical and functional observations, body weight, food and water consumption, haematology and clinical chemistry, macroscopy, organ weights and histopatology were found. Incidental findings reported were within historical control values and without a dose-response relationship. Therfore the NOAEL for parental toxiciy is considered 1000 mg/kg bw.

Examination of reproductive performance (pregnancy rate, mating index, pre-coital interval, gestation length, number of corpora lutea/implantation sites, implantation loss) did not show any adverse effects of the test substance. No adverse effects on the number, sex, body weight, clinical signs of the offspring was reported. Therefore it is concluded tht the NOAEL for reproduction is 1000 mg/kg bw.