Tall oil, maleated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 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:

Fatty acids, C14-18 and C16-18-unsatd., maleated

IUPAC Name:

Fatty acids, C14-18 and C16-18-unsatd., maleated

Test material form:

other: liquid (semi-solid or wax-like at 20°C)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Wistar Crl:WI rats- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld- Age at study initiation: 11 weeks- Weight at study initiation: males: Males: 350 g – 382 g, Females: 183 g - 218 g- Fasting period before study: overnight prior to treatment- Housing: 5 animals of the same sex and group/cage with the exception of the mating and gestation/delivery period, when theywere paired or individually housed, respectively. (cage: Type II and/or III polycarbonate)- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: at least 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20,1-25- Humidity (%): 36-70- Air changes (per hr):15-20- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

VEHICLE- Concentration in vehicle: 20, 60, 200 mg/ml- Amount of vehicle (if gavage): 5 mL/kg bwThe test material and the vehicle was warmed up on a water bath to 50 C for approximately 10 minutes separately and mixed up on a hot stirrer plate. Once a suitable formulation was obtained, the container was removed from the plate. Pending administration to the animals, the dose formulations were stirred on a magnetic stirrer at room temperature and were protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of WS400104 formulations for concentration and homogeneity was performed using validated HPLC method (CiToxLAB study code 11/352-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400104 from propylene glycol ranged between 92% and 106% (92% - 104%, 97% - 106%, 96% - 102% respectively to the three analysis performances).

Details on mating procedure:

- M/F ratio per cage: 1/1- Length of cohabitation: until copulation occurred, for up to 7 days.- 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.- Mating of siblings was avoided.

Duration of treatment / exposure:

Main males: 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week)Main females: ca. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til Day 4 post-partum (PPD4))Satellite females (not mated): 35 daysOffspring were not dosed (up to PPD4).

Frequency of treatment:

daily, 7 days/week

Duration of test:

Main males: 35 days Main females: ca. 47 days (necropsy on PPD5)Offspring: from birth to PND4

No. of animals per sex per dose:

12 animals /sex / doseSatellite females: 5 / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose finding toxicity study with WS400104 (administered via oral gavage to Wistar rats for 7 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw) showed no overt adverse effects related to the test material (7.5.1 Dose Range Finding Study 7 days_WS400104).Based on these results, the dose levels selected for the main study were 0, 100, 300 and 1000 mg/kg bw/day.This study (OECD Guideline 422) was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study . Therefore, animals initially entering the study were divided into toxicity subgroup animals (Satellite females) and reproductive subgroup animals (Main females and males), whereby 5 of the 12 Main males (used for pairing) per dose group formed the toxicity male Subgroup A.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: twice dailyAll animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.Delivery process was observed as carefully as possible. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: at least weekly, observations in a standard arenaBODY WEIGHT: Yes - Time schedule: Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination). SACRIFICE- Maternal animals: All surviving animals on Day 5 of lactation (PND5).GROSS NECROPSY- Gross necropsy consisted of external examinations.- Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the Main females as applicable.ORGAN WEIGHTSWeight of the following organs of all adult animals were determined:- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain- With a precision of 0.001 g: ovaries, pituitaryFOOD CONSUMPTION: yes-Time schedule: at least weeklyHISTOPATHOLOGY :Detailed histological examinations was performed in all female adults of control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.Other examinations: yes, clinical chemistry , haematology and urinalysis in the toxicity groups (satellite females and subgroup A - males), see: Combined repeated dose toxicity_gavage_rat_WS400104

Ovaries and uterine content:

The ovaries and uterine content was examined after termination of the dams on Day 5 post-partumExaminations included:- Number of corpora lutea: Yes - Number of implantations: Yes Post implantation survival was determined.In addition, gestation length (time elapsing between detection of mating and commencement of parturition) was recorded.

Fetal examinations:

- External examinations: from PPD0 to PPD4, all pubs per litterparameters observed: number and sex of pups, stillbirths, live births; postnatal mortality; presence of gross anomalies, weight gain (PPD0-PPD4), physical or behavioural abnormalities

Statistics:

Performance with the statistical program package SPSS PC+4.0.The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test,the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Indices:

Female Mating Index: Number of sperm-positive females : Total Number of females cohabited x 100 Female Fertility Index: Number of pregnant females : Number of sperm-positive females x 100Gestation Index: Number of females with live born pups : Number of pregnant females x 100Formulas for Calculation of Pups’ Mortality and Sex Ratio IndicesSurvival Index:Number of live pups (at designated time) : Number of pups born x 100Pre-implantation mortality: (Number of Corpora lutea − Number of Implantations) : Number of Corpora lutea x 100Intrauterine mortality: (Number of implantations - Number of liveborns) : Number of implantations x 100Total mortality: (Number of implantations - Number of viable pups (d4)) : Number of implantations x 100Post-natal mortality: (Number of viable pups (d0) - Number of viable pups (d4)) : Number of viable pups (d0) x 100Sex ratio: (Number of pups examined − Number of males) : Number of pups examined x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: In the high dose group (1000 mg/kg bw/day) local irritation of the pars non-glandularis of the stomach was found.Details on maternal toxic effects:Minimal/mild hyper/paraceratosis of the nonglandular gastric mucosa in stomach was observed in 2 of 5 females after administration of 1000 mg/kg bw/day. No systemic effects were observed.No adverse effect was found on reproduction / developmental parameters at this dose level.There were no effects of treatment noted during gestation, parturition or the post-partalperiod, neither histological findings in the reproductive organs. See for details: 7.5.1 Combined repeated dose toxicity_gavage_rat_WS400104

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:WS400104 administered to parental generation at up to 1000 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day.The sex ratios were similar in the Control and treated groups.There was no effect of treatment on the offspring body weight or body weight gain.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

other: No adverse effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No adverse effects

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Daily oral gavage administration of fatty acids, C14-18 and C16-18-unsatd., maleated at dose levels of 100, 300 and 1000 mg/kg day was not associated with signs of developmental toxicity and the NOAEL was considered to be 1000 mg/kg /day.

Executive summary:

In a key combined repeat dose reproductive/developmental toxicity study, the test material (Fatty acids, C14-18 and C16-18-unsatd., maleated) was administered daily via oral gavage at doses of 0, 100, 300, and 1000 mg/kg bw/day to Wistar rats (12/sex/dose) in a propylene glycol vehicle. Male rats were treated for a period of 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week) while female rats were treated for approximately 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til PPD 4). A satellite group of nulliparous and nonpregnant female rats (5/sex/dose) was also employed in the study and treated with the test material for a period of 35 days.

All animals were observed twice daily for signs of clinical toxicity with special attention directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Body weight determinations were made for adult main and satellite animals on Day 0, at least once weekly thereafter, and at termination. Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on post partal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination). Food consumption was determined once weekly (at least) and hematology, urinalysis, and clinical chemistry parameters also evaluated (prior to necroscopy) on Day 35. Neurobehavioral examinations were undertaken in the study during the last exposure week (Day 32). Parameters such as testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating gland were examined in male parental generations, all males (12/dose). For the F1-offspring, the number and sex of pups, stillbirths, live births; postnatal mortality; presence of gross anomalies, weight gain (PPD0-PPD4), physical or behavioural abnormalities were observed for in the study. Necroscopy was conducted at termination on Day 35, i.e. one day after the last treatment following overnight period food deprivation, or PND5. Organ weights of uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain, and pituitary were also determined.

 

Statistical evaluation of data was carried out in this study using SPSS PC+4.0 (SPSS Hungary Kft, Budapest). Additionally, mating and fertility Indices, as well as mortality and sex ratio indices were calculated.

No signs of mortality or clinical toxicity were observed through the study period. Body weight and food consumption remained unaffected post-treatment with the test material. There was no effect of treatment on the oestrus cycle or reproductive parameters. There was no effect of treatment noted during gestation, parturition or the post-partal period. Test material related microscopic findings were found at 1000 mg/kg bw/day (High dose) in stomach, in the form of hyper/paraceratosis of the nonglandular gastric mucosa. This minimal to mild multifocal change occurred in 4 of 5 males and 2 of 5 females. There was no evidence of test item-related histological findings in the reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were revealed normal histological pictures. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.

There was/were no mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. In single pups at 100, 300 and 1000 mg/kg haemorrhage was observed on PND0. The sex ratios were observed to be similar in the Control and treated groups. Body weight or body weight gain remained unaffected by treatment with fatty acids, C14-18 and C16-18-unsatd., maleated.

Daily oral gavage administration of fatty acids, C14-18 and C16-18-unsatd., maleated at dose levels of 100, 300 and 1000 mg/kg day was not associated with signs of developmental toxicity and the NOAEL was considered to be 1000 mg/kg /day.