Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

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:

21 March 2014 to November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed to a recognised guideline and used GLP. The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation. Deviations from the target range for relative humidity were less than ideal, overall it is considered that these deviations had no adverse impact on the scientific purpose of the study.

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:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Deviation No.1 Multi-Site Study Details The Test Site Quality Assurance for histology processing ; Deviation No. 2 Animal Husbandry Environment The Study Plan target values for temperature and relative humidity controls were 22 ± 3ºC and 50 ± 20%

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

EC Number:

940-875-4

Molecular formula:

C3H8O3 - C99H182O20

IUPAC Name:

Esterification products of dihydrofuran-2,5-dione, C16-24 (even numbered) alkenyl with propane-1,2,3-triol and propane-1,2,3-triol oligomers

Test material form:

other: viscous amber liquid

Details on test material:

- Physical State/Appearance : Amber colored extremely viscous liquid
- Purity : Not supplied
- Date Received : 08 August 2013
- Storage Conditions : Room temperature in the dark
- Expiry Date : 08 August 2014
No correction for purity was made.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks
- Weight at study initiation: Males: 240 to 311 g ; Females: 164 to 211 g
- Housing: Animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from
Study Plan.
-The animals were allowed free access to food and water. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 15 cycles/h of air
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 1-14 days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- Pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The formulation investigated during the study were found to comprise test item in the range of 91% to 108% and, thus, the required content of +/- 10% with reference to the nominal content was met.
- The test item was found to be stable in the formulations when kept 21 days in the refrigerator (4°C) due to results which met the variation limit of 10% from the time-zero mean.
- In conclusion, the results indicate the accurate use of the test item and poluethylement glycol 400 as vehicle for this study. The formulations were found to be homogeneously prepared abd sufficient formaultion stability storage was proven.

Duration of treatment / exposure:

Males:
- 2 weeks before mating, during the mating (maximum of 2 weeks) and post-mating periods (at least 2 weeks) until sacrifice (at least 6 weeks in total).

Females:
- 2 weeks before mating, during the mating period (maximum of 2 weeks), during pregnancy and lactation, until Day 4 post-partum inclusive, (or until sacrifice for un-mated and non-pregnant females and females which did not deliver) then up to 8 weeks.

Frequency of treatment:

Once daily, 7 days a week

Details on study schedule:

Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
The male dose groups were killed and examined macroscopically on Day 43.
At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Remarks:

Doses / Concentrations:
Males& Females: dose levels 100, 250, 500 and 1000 mg/kg bw/day of active content
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
Time schedule:
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
Time schedule for examinations:
- Male: On the first day of treatment, prior to dosing (Day 1), then once a week until sacrifice.
- Female: Once daily until mated (or until sacrifice) and on Days 0, 7, 14 and 20 p.c. and Days 1 and 45 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Time schedule:
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing.
- Male: Once a week, over a 7-day period, from the first day of treatment until sacrifice.
- Female: For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4)..
- During the mating period, the food consumption was noted for neither males nor females.
- Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated for females during gestation and lactation.
- Water intake was observed daily by visual inspection of water bottles for any overt changes.

MAITING
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

PREGNANCY AND PARTURITION:
- Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends. The length of gestation and the partiurition Index were calculated.
For each female is noted: date of pairing, date of mating, date and time of observed start of parturition; date and time of observed completion of parturition/

Oestrous cyclicity (parental animals):

No

Sperm parameters (parental animals):

Parameters examined in male animals:
- Testes and epididymis weight were recorded.

Litter observations:

LITTER SIZE
- Total litter size and numbers of pups were recorded as soon as possible after birth.
- Litters were observed daily for recording of the number of live, dead on Day 1 and 4 post partum.
- External examination of each pup was carried out and any external malformation was noted.
- Sex of offspring on Days 1 and 4 post partum

CLINICAL SIGNS
- Clinical condition of offspring from birth to Day 5 post partum.
- All live offspring were assessed for surface righting reflex on Day 1 post partum.

BODY WEIGHT
- Weight of each pup was recorded on Days 1 and 4 post-partum.

Postmortem examinations (parental animals):

SACRIFICE: Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

GROSS NECROPSY
- All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded
- In all females, the number of implantation sites and corpora lutea was recorded.

ORGAN WEIGTHS
- The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.
- Ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY
- Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Coagulating gland, Prostate, Epididymides, Seminal vesicles, Ovaries Testes, Mammary gland (females only) Uterus/Cervix, Pituitary, Vagina and for all animals gross lesions

Postmortem examinations (offspring):

SACRIFICE
- Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM 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 analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed 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 analyzed 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 Mann- Whitney U test (non-parametric).
Data not analyzed by the Provantis data capture system was assessed separately using the R Environment for Statistical Computing.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Reproductive indices:

- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating
- Mating index: [Number of mated animals / Number of paired animals] X 100
- Pregnancy Index(%) : [Number of pregnant female / Number of mated pairs] X 100
- Gestation index: [Number of females with live born pups / Number of pregnant females] X 100
- Pre–implantation loss (%): [Number of corpora lutea - Number of implantation sites]/[Number of corpora lutea] x 100
- Post–implantation loss (%): [Number of implantation sites - Total number of offspring born]/[Number of implantation sites] x 100
- Parturition Index (%): [Number of females delivering live offspring / Number of pregnant females] X 100

Offspring viability indices:

- Live birth index: [Number of live born pups / Number of delivered pups] X 100
- Viability Index (%): [Number of offspring alive on Day 4] / [Number of offspring alive on Day 1] X 100
- Sex ratio (% males): [Number of male offspring]/ [Total Number of offspring] 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:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No mortality and no test item treatment-related clinical signs were noted during the study.
- No toxicologically significant clinical observations were detected in treated animals.
- Episodes of increased salivation were evident in animals of either sex treated with 1000 and 500 mg/kg bw/day from Day 22 onwards. One male treated with 500 mg/kg bw/day also showed noisy respiration on Day 36 only. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in the absence of any associated changes were considered of no toxicological significance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- There were no treatment-related effects in body weight development at 100, 250, 500 or 1000 mg/kg bw/day.
- Females treated with 250 mg/kg bw/day showed a statistically significant increase in body weight on Day 4 of lactation. An increase in body weight is not representative of an adverse effect of treatment.
- No adverse effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study. Statistical analysis of the gestation and lactation data did not reveal any significant intergroup differences.
- No adverse effect on water consumption was detected in treated animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating data:
- There were no treatment-related effects on mating performance.
Fertility data:
- No treatment-related effects on fertility were detected for treated animals, when compared to controls.
Two control females and two females treated with 1000 mg/kg bw/day did not achieve pregnancy following evidence of mating. A further female treated with 1000 mg/kg bw/day showed corpora lutea and implantation sites however did not give birth to any live offspring.
The male partner of one of the non pregnant females treated with 1000 mg/kg bw/day had small epididymides and small and flaccid testes. Histopathological examination of this male revealed testicular tubular atrophy and reduced sperm in the epididymides and this was considered to be the cause of the non pregnancy. The remaining occurrences were considered incidental.

DATA:
No significant differences were detected for corpora lutea, implantation counts, pre or post implantation losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males:
- No treatment-related effects were evident in the organ weights measured.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- No toxicologically significant macroscopic abnormalities were detected.
One male treated with 1000 mg/kg bw/day had small epididymides and small and flaccid testes. This male partnered a non pregnant female. Histopathological examinations revealed testicular tubular atrophy and reduced sperm in the epididymides. In isolation this was considered to be incidental and of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- No histopathological findings related to the test item treatment were observed.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: The oral administration of the substance to rats by gavage, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects.

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)
- Live birth index was 100 % in each group and viability index on Day 4 post-partum was similar in the control, 50 and 450 mg/kg bw/day groups.
- At 150 mg/kg bw/day, viability index was slightly lower (88 % vs. 96 % in controls) as the number of pups found dead or cannibalized between Days 1 and 4 post-partum was statistically significantly higher than controls (13 vs. 6, p<0.05). In the absence of a dose-relationship, this variation was not attributed to treatment with the test item.

CLINICAL SIGNS (OFFSPRING)
- No test item treatment-related clinical signs were noted in pups.

BODY WEIGHT (OFFSPRING)
- Mean pup body weights and body weight gains were similar to those of the controls throughout the lactation period.

SEX RATIO (OFFSPRING)
- There were no toxicologically significant differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
- No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

OFFSPRING GROWTH AND DEVELOPMENT
There were no toxicologically significant effects detected.
Male and female offspring weights on Day 4 post partum and body weight gain throughout lactation were statistically significantly increased in litters from females treated with 1000 mg/kg bw/day when compared to control offspring. An increase in offspring body weight development is not considered to represent an adverse effect of treatment therefore the intergroup differenceswere considered not to be of toxicological importance. Statistical analysis of the litter weights or surface righting reflex data did not reveal any significant intergroup differences.
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small, cold, no milk in stomach, found dead or missing were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: The oral administration of the substance to rats by gavage, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of the substance to rats by gavage, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to be compatible with 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 four groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week prepairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 250, 500 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, body weight change, dietary intake 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. Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

 

Results

…….

Adult Responses

Mortality

There were no unscheduled deaths.

Clinical Observations

No toxicologically significant clinical observations were detected in treated animals.

Body Weight

There were no treatment-related effects in body weight development at 100, 250, 500 or

1000 mg/kg bw/day.

Food Consumption

No adverse effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study.

Water Consumption

No adverse effect on water consumption was detected in treated animals.

 

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

Fertility as assessed by pregnancy rate was unaffected by treatment at all dose levels.

Gestation Length

There were no differences in gestation lengths. The distribution for treated females was compared to controls. Gestation lengths were between 22 and 23½ days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4post partumwere comparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4post partumwere comparable to controls. Surface righting was also comparable to controls.

Offspring Observations

No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology

Necropsy

No toxicologically significant macroscopic abnormalities were detected.

Organ Weights

No treatment-related effects were evident in the organ weights measured.

Histopathology

There were no treatment-related microscopic abnormalities detected.

 

Conclusion

The oral administration of the substance to rats by gavage, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects.