Fatty acids, tall-oil, C12-15-alkyl esters, sulfated, sodium salts

Toxicity to reproduction

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:

31 March 2010 to 19 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study (OECD 422)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: other guideline: unspecified EC No. 440/2008 guidelines

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Rape oil, bisulfited, sodium salt

EC Number:

281-975-1

EC Name:

Rape oil, bisulfited, sodium salt

Cas Number:

84082-27-9

IUPAC Name:

84082-27-9

Details on test material:

- Name of test material (as cited in study report): FLL sample 4
- Physical state: viscous liquid
- Analytical purity: 90%
- Impurities (identity and concentrations): 10% water by Karl-Fischer
- Lot/batch No.: 240210
- Expiration date of the lot/batch: not reported
- Storage condition of test material: room temperature in the dark
- Other: yellow/brown color
- All other template details: Not reported

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 12 weeks old
- Weight at study initiation: males weighed 304-375 g, females weighed 189-234 g
- Fasting period before study: not reported
- Housing: initially housed in groups of 5 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper with one male and one female. Following successful mating, males were returned to their original cages while mated females were housed individually during gestation and lactation in solid floor cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum access to a pelleted diet of Rodent 2018C Teklad Global Certified Diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITION
S- Temperature (°C): 21 +/- 2 degrees C
- Humidity (%): 55 +/- 15 %- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 14 April 2010 To: 29 May 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

(400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: formulations were prepared twice monthly and stored at approximately 4 degrees C in the dark

DIET PREPARATION: Not applicable, dosing was via oral gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water): This vehicle was considered to be the most suitable vehicle to provide a homogeneous and stable formulation following a vehicle trial performed in the preliminary study. The formulations were stable for at least 21 days.
- Concentration in vehicle: 0, 37.5, 87.5, 250 mg/ml
- Amount of vehicle (if gavage): up to 4 ml/kg
- Lot/batch no. (if required): not reported
- Purity: not reported :

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of successful mating
- Proof of pregnancy: not reported
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable
- Further matings after two unsuccessful attempts: not applicable, only one male:female mating attempt
- After successful mating each pregnant female was caged (how): individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Any other deviations from standard protocol: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of FLL Sample 3 in the test item formulations was determined by gel permeation chromatography (GPC) using an external standard technique. The standard and sample solutions were analysed by GPC using the following conditions: HPLC System : Agilent Technologies 1050, incorporating autosampler and workstation; Column: PL Gel HTS-D (150 x 7.5 mm id); Column temperature: 40 degrees C; Mobile phase : Tetrahydrofuran; Flow-rate : 1 ml/minDetector : UV at 230 nm; Injection volume : 100 μl; Retention time : ~ 3.9 mins.
The concentration of the test item measured (as % of nominal) ranged from 88 to 104 % of nominal concentrations of 37.5 to 250 mg/ml. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

males: 42 days
females: 42-47 days

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 14 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 350, 500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a preliminary study was performed with three groups of 6 Wistar rats (3 male and 3 female), with the test item administered by oral gavage for fourteen consecutive days at dose levels of 250, 500, and 1000 mg/kg/day. In all of the dose levels, there was no mortality, no adverse effects on body weight change, no macroscopic abnormalities detected upon necropsy, and no adverse effects on dietary or water intake.
- Rationale for animal assignment (if not random): used a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups

Positive control:

Not reported

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week
- Cage side observations included: overt signs of toxicity, ill-health and behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of treatment and at weekly intervals thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, although the test item was not dosed in the water
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data, The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, although the test item was not dosed in the water
- Time schedule for examinations: daily

Oestrous cyclicity (parental animals):

Not reported

Sperm parameters (parental animals):

Parameters examined in all males: testis weight, epididymides weight, seminal vesicles and prostate weights

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, haematological and blood chemicals measured

GROSS EXAMINATION OF DEAD PUPS: not applicable, no pups died before the end of the study

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed following completion of female gestation and lactation phases.
- Maternal animals: All surviving animals were sacrificed on Day 5 post partum.

GROSS NECROPSY
- Gross necropsy consisted of full external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
See Table 4 for tissues removed and examined. Organ weights were obtained for adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary (post-fixation), protate, seminal vesicles, spleen, testes, thymus, thyroid, uterus.

Postmortem examinations (offspring):

SACRIFICE
- All offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of full external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
See Table 4 for tissues removed and examined. Organ weights were obtained for adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary (post-fixation), protate, seminal vesicles, spleen, testes, thymus, thyroid, uterus.

Statistics:

Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Reproductive indices:

Fertility indices (%): mating index and pregnancy index
Parturition index (%), implantation loss (%), live birth indes (%), viability index (%), sex ratio (% males)

Offspring viability indices:

= number of offspring alive on Day 4 / number of offspring alive on Day 1 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

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: One male treated with 350 mg/kg/day was found dead on Day 25, but this death was confirmed to be attributed to inappropriate dosing technique and not related to test material toxicity. Other clinical observations included post-dose increased salivation, noisy respiration, and were considered unrelated to test item toxicity.

BODY WEIGHT AND WEIGHT GAIN: No differences in body weight change were evident for females during the pre-mating, gestation or lactation phases of the study. No adverse effects on body weight change were detected for treated males when compared to controls during the treatment period.

FOOD EFFICIENCY, FOOD AND WATER CONSUMPTION: no treatment-related intergroup differences

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no significant treatment-related differences in mating performance, fertility, gestation length, litter size or viability. There was one non-pregnant female from the control group.

HAEMATOLOGY: no treatment related effects detected in the haematological parameters measured

CLINICAL CHEMISTRY: There were no toxicologically significant effects detected in the blood chemical parameters measured. Females treated with 1000 and 350 mg/kg/day showed a statistically significant reduction in lymphocyte counts, when compared to control values. The significance achieved was minimal (P<0.05), and all individual values for these animals were within the normally expected ranges. These minor reductions were therefore considered to be attributable to one higher than expected control value, and were unrelated to treatment with the test item.

NEUROBEHAVIOUR: There were no treatment-related changes in sensory reactivity or functional performance.

ORGAN WEIGHTS: No toxicologically significant effects were detected in the organ weights measured. Males treated at all dose levels showed statistically significant increases in absolute and body weight-relative pituitary weights when compared to controls. In the absence of a convincing dose-related response or any histopathological changes to suggest an effect of treatment, these increases were considered not to represent test item toxicity.

GROSS PATHOLOGY: There were no toxicologically significant macroscopic abnormalities detected. The interim death male treated at 350 mg/kg/day showed reddened lungs and a fluid-filled thoracic cavity. These findings are associated with inappropriate administration of the test item resulting in death, and are unrelated to test item toxicity.
The remaining macroscopic findings consisted of reddened lungs for one female treated with 350 mg/kg/day and an irregular surface edge of the liver was observed for another female treated with 350 mg/kg/day. Hydronephrosis of the right kidney was evident for one male treated with 150 mg/kg/day. These findings were isolated to one animal on each occasion and in the absence of any histopathological correlations, were considered to have arisen incidentally, with no relation to treatment with the test item. Epithelial sloughing of the glandular region of the stomach, hydronephrosis of the left kidney and ovaries encased in a fluid-filled sac were observed in the female control group. These findings are occasionally observed in repeated dose studies of this type, and in the absence of treatment in this group, the findings were purely incidental in nature.

HISTOPATHOLOGY: There were no treatment related microscopic abnormalities detected in terminal kill animals. Marked necrosis of the trachea was evident for the interim death male. The cause of death for this animal was therefore confirmed to be attributable to inappropriate dosing technique and was unrelated to treatment. All other findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age or findings associated with the status post partum.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects, no treatment-related effects were observed for any of the reproductive endpoints measured

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:

no effects observed

Details on results (F1)

Offspring Litter Size and Viability
No treatment-related differences in corpora lutea, implantation sites, or pre- and postimplantation losses were detected for females from treated groups when compared to controls. No differences in litter size or viability were evident for offspring from treated litters when compared to those from the controls. Statistical analysis of these data did not result in any significant intergroup differences.

Offspring Growth and Development
There were no differences in litter weights or mean offspring body weights between control and treated animals.
No treatment-related effects in litter weights, offspring weights or surface righting were detected for litters from treated groups when compared to control litters. Statistical analysis of these data did not result in any significant intergroup differences.
The number of interim offspring found dead or missing (cannabalised by the mother) was essentially similar throughout the control and treatment groups. No treatment-related clinical signs were observed. The clinical signs observed were low incidence findings commonly observed in reproductive studies of this type and unrelated to test item toxicity.

Necropsy
No treatment-related macroscopic abnormalities were detected for the one interim death offspring from the control group, or for remaining offspring at termination on Day 5 post partum. The incidental findings observed in the terminal kill offspring (small size, hydronephrosis of the kidneys, wound to the head) were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Histopathology
There were no treatment related microscopic abnormalities detected in terminal kill animals.
All findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age and were unrelated to treatment.

Effect levels (F1)

Dose descriptor:

NOEL

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: overall effects, no treatment-related effects were observed for any of the reproductive endpoints measured

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:

No treatment-related effects were observed for reproduction, therefore, a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.

Executive summary:

Study Summary:

Introduction. The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and meets the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

This study was also designed to meet the requirements of Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods. The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 150, 350 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum. Haematology and blood chemistry were evaluated prior to termination on five males and five females from each dose group (due to a technical error, only four males were assessed from the 350 mg/kg/day dose group).

Surviving males were terminated on Day 43, followed by the termination of all females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Adult Responses:

Mortality. There were no unscheduled deaths attributable to test item toxicity.

 

Clinical Observations. No clinically observable signs of toxicity were detected.

 

Functional Performance Tests. No treatment-related effects were detected.

 

Body Weight. No adverse effects on body weight change were detected.

 

Food Consumption. No adverse effects on dietary intake were detected.

 

Water Consumption. Daily visual inspection of water bottles did not reveal any significant intergroup differences.

 

Reproductive Performance:

Mating. No treatment-related effects were detected in mating performance.

 

Fertility. No treatment-related effects on fertility were detected.

 

Gestation Lengths. No treatment-related effects were detected in gestation lengths.

 

Litter Responses:

 

Offspring Litter Size and Viability. No treatment-related effects were detected.

 

Offspring Growth and Development. No treatment-related effects were detected.

 

Laboratory Investigations:

Haematology. No treatment-related effects were detected.

 

Blood Chemistry. No toxicologically significant intergroup differences were detected.

 

Pathology:

Organ Weights. No toxicologically significant effects of treatment were detected.

 

Necropsy. No treatment-related macroscopic abnormalities were detected.

 

Histopathology. No treatment-related microscopic abnormalities were detected.

 

Conclusion. The oral administration of FLL Sample 4 to rats by gavage at a maximum dose level of 1000 mg/kg/day did not result in any toxicologically significant effects of treatment. The minor effects detected in this study were not considered to represent an adverse effect of treatment; therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 1000 mg/kg/day for systemic toxicity.

 

No treatment-related effects were detected for reproductive performance, hence a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.