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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

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 (incl. QA statement)

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

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
- Sex: Males & Females
- 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.

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.

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

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

Duration of test:

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

Details on study design:

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.

Examinations

Maternal examinations:

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

Ovaries and uterine content:

No

Fetal examinations:

No

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)

Indices:

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

Historical control data:

No

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no unscheduled deaths.
No toxicologically significant clinical observations were detected in treated animals.
There were no treatment-related effects in body weight development at 100, 250, 500 or 1000 mg/kg bw/day.
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.
No adverse effect on water consumption was detected in treated animals.
No treatment-related effects were detected in mating performance.
Fertility as assessed by pregnancy rate was unaffected by treatment at all dose levels.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental 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.