DL-hexane-1,2-diol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from January 18,2006 to May 10, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test following GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

DL-hexane-1,2-diol

EC Number:

230-029-6

EC Name:

DL-hexane-1,2-diol

Cas Number:

6920-22-5

Molecular formula:

C6H14O2

IUPAC Name:

hexane-1,2-diol

Test material form:

other: clear colorless liquid

Details on test material:

CAS No.: 6920-22-5
EC No.: 230-029-6

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandly:
A total of ninety-six time-mated female Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches of forty-eight animals two days apart; each delivery batch contained twenty-four animals at Day 1 of gestation and twenty-four animals at Day 0 of gestation. All females were supplied with stud male identification records and were identified using the supplier’s standard identification methods.
Throughout the study the animals were given pelleted diet (Certified Rodent diet PMI 5002 supplied by BCM IPS Ltd., London UK) and water was supplied via bottles attached to each cage, ad libitum. The diet and water were considered not to have contained any contaminants at a level which may have affected the outcome of this study.
Upon arrival the females were temporarily housed in groups of four or five in polypropylene cages with stainless steel grid floors and tops. Animals were then weighed and allocated to the study. Following allocation the females were caged individually in polypropylene cages with solid floors and stainless steel grid tops. Softwood chips were used as bedding material.
The animals were housed in a single air-conditioned room, providing at least fifteen air changes per hour. The room was maintained to operate within a temperature range of 19 and 23 °C and a relative humidity of 40 and 70% with these conditions monitored on a daily basis. The lighting within the room was controlled to allow twelve hours of continuous light within a twenty-four hour period.
Allocation:
On the day of arrival the females were assigned to treatment groups using a randomisation procedure based on stratified bodyweight to ensure, as far as possible, similar group mean bodyweights for each treatment group. The females were assigned to positions on the cage battery using a randomised block design.
At allocation females were given a unique earmark for the study according to the assigned number for each dose group. Each cage was identified with a colour coded cage card containing information including project number, dose group and animal number.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Experimental Preparation
The test material was prepared weekly as a solution in distilled water (vehicle) by weighing an appropriate amount of test material into a suitable container and adding sufficient vehicle to make the required volume. The test material formulations were shaken until all the test material dissolved. Formulations were therefore prepared weekly and stored at approximately +4 °C in the dark.

Dosing
Animals were dosed with the appropriate concentration of dose formulation once daily, using a metal catheter and syringe between Days 5 and 19 of gestation. Control females received vehicle only. The dose administered was adjusted for most recent bodyweight using a constant dose volume of 5 ml/kg bodyweight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Results showed the formulations to be stable for at least fourteen days.

Details on mating procedure:

The day that positive evidence of mating was observed was designated Day 0 of gestation.

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Duration of test:

20 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 30, 100, 300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Selection of Dose Levels
Dose levels of 300, 100 and 30 mg/kg/day were selected for use on this study based on results of a preliminary oral gavage prenatal developmental toxicity study in the rat (SPL Project Number 2082/0014). In this preliminary study, a dose level of 500 mg/kg/day was associated with adverse clinical signs that excluded this dosage level from use in this main investigation. A high dosage of 300 mg/kg/day was therefore chosen in anticipation of a degree of toxicity that would not impair the assessment of embryofoetal development. Lower dosages represent approximately three-fold reductions from this high dosage

Examinations

Maternal examinations:

1. Morbidity/Mortality
All females were checked twice daily during the normal working week and once daily at weekends.
2. Clinical Observations
All females were observed once daily, in the morning throughout gestation and, additionally, one hour after dosing, throughout the dosing period, for clinical signs of toxicity.
3. Bodyweight
All females were weighed on Days 3, 5, 6, 7, 8, 11, 14, 17 and 20 of gestation.
4. Food Consumption
Food consumption for individual animals was recorded for discrete periods throughout the study on Day 3 to 5 Day 5 to 8 Day 8 to 11 (or 9 to 11) Day 11 to 14, Day 14 to 17 and Day 17 to 20 of gestation.

Ovaries and uterine content:

Females were killed on Day 20 of gestation by carbon dioxide asphyxiation followed by cervical dislocation. Each animal was examined externally and internally for macroscopic abnormalities. The ovaries and uteri of pregnant females were removed, examined and the following data recorded;
i) Pregnancy status
ii) Number of corpora lutea
iii) Gravid Uterus weight
iv) Number, position and type of intra-uterine implantation
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue.
Late Death: Separate embryonic/foetal and placental tissue visible.
Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes.

Fetal examinations:

The foetuses were killed by subcutaneous injection of sodium pentobarbitone. Except where conditions dictate otherwise, alternative foetuses were identified using an indelible marker and placed in Bouin’s fixative. These foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red. The foetuses were examined for skeletal development and anomalies.
All implantations and viable foetus were numbered according to their intra-uterine position as follows (V = viable foetuses):
Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
VI V2 V3 V4 V5 V6 V7 V8 V9 V10 Vll V12 V13 V14 V15 V16
v) Foetal sex
vi) External foetal appearance
vii) Foetal weight
viii) Placental weight

Statistics:

Data were processed to give litter mean values, group mean values and standard deviations (where appropriate). The litter was regarded as the standard unit of assessment therefore values were first calculated within each litter and group mean values were calculated as the mean of these litter values.
The following parameters were analysed statistically, where appropriate, using the test methods outlined below:
Bodyweight, bodyweight change and food consumption: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnet’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
Litter data and litter, placental and foetal weights: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control value against treated values.

Indices:

Percentage pre-implantation loss was calculated as:
(Number of corpora lutea - number of implantations)/ number of corpora lutea
Percentage post-implantation loss was calculated as:
(Number of implantations - number of live foetuses)/ number of implantations

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
1 Clinical Observations and Mortality
There were no clinical signs associated with treatment and all females survived to scheduled termination on Day 20 of gestation.
2 Bodyweight
No adverse effects of treatment on bodyweight or bodyweight gain were apparent throughout the study at 300, 100 or 30 mg/kg/day
3 Food Consumption
No adverse effects of treatment on food consumption were apparent throughout the study at 300, 100 or 30 mg/kg/day,
4 Necropsy
All animals were pregnant and no macroscopic abnormalities were observed at macroscopic post mortem examination on Day 20 of gestation.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Data and Litter Placental and Foetal Weights
There was no obvious adverse effect of maternal treatment on in-utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation loss at 300, 100 or 30 mg/kg/day. Sex ratio was essentially similar in all groups and did not indicate any selective effect on survival for either sex.
Intergroup differences in mean litter, placental and foetal weights did not indicate any obvious effect on embryofoetal growth at these dosages.
2. Foetal examination
Neither the type, distribution nor incidence of foetal findings observed during external examination at necropsy or subsequent detailed visceral and skeletal examinations indicated any adverse effect of maternal treatment on foetal growth or development at 300, 100 or 30 mg/kg/day.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results given in this study, test article caused no adverse effect on the pregnant rat on the developing conceptus at dose levels up to 300 mg/kg. The ‘no-observed adverse effect level’(NOAEL) was therefore considered to be 300 mg/kg/day.

Executive summary:

This study was conducted following OECD guideline 414 and GLP regulations to investigate the effects of test article on the embryotoxic/teratogenic development of rat. A total of 96 time-mated female Crl:CD(SD) IGS BR strain rats were administered orally, once daily, by gavage, at dose levels of 0, 30, 100 and 300 mg/kg/day. Clinical signs, bodyweights and food consumption were recorded during the study. The females were killed on Day 20 of gestation and subjected to macroscopic necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Treatment at dosage up to 300 mg/kg/day was well tolerated by the pregnant females and was not associated with any effects on clinical condition, bodyweight change, food intake or necropsy observations. There were no effects at dosages up to 300 mg/kg/day on embryofoetal survival, growth and development.

The 'No- observed-adverse-effect-level' (NOAEL) for the pregnant female and for embryofoetal survival, growth and development was therefore considered to be 300 mg/kg/day.