Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 January 2013 to 15 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A pre-natal developmental toxicity study (OECD 414) was completed in accordance with test guidelines and the principles of GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
- Name of test material (as cited in study report): Trimethylolpropane diallyl ether-90 (TMPDE-90). 2,2-bis(allyloxymethyl) butan-1-ol

- Physical state: colourless liquid
- Analytical purity: 92.3% (dose calculations were corrected for the purity value - correction value 1.083)

- Lot/batch No.: 4252001
- Expiration date of the lot/batch: 1 August 2013

- Stability under test conditions: stable for duration of study at ambient laboratory environmental conditions
- Storage condition of test material: ambient, protected from light and sealed under an inert gas

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sprague-Dawley rats obtained from Charles River, Germany on 11 January 2013 or 8 March 2013
- Age at study initiation: Time-mated females were circa 9 weeks old at time of dosing
- Weight at study initiation: 197-346g (this deviated from the protocol range of 200-240g, but the animals were within the age range specified in the protocol). A second batch of animals were supplied on 8 March, circa 9 weeks old.
- Fasting period before study: No
- Housing: suspended polycarbonate cages with wood shavings for bedding. Individually housed
- Diet (e.g. ad libitum): Rat and Mouse modified No 3 Diet SQC expanded
- Water (e.g. ad libitum): ad libitum access to tap water supplied in water bottles
- Acclimation period:3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 January 2013 To: 26 March 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose levels of 0, 50, 200 or 800 mg/kg bw/day. Dose formulations were stirred continuously during dose administration. Dose levels and dose preparations were selected following a review of previous study results.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 10, 40, 160 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were sampled during week 1 and 2. Analyses were completed using gas chromatography with flame ionisation detection.
Samples were analysed for achieved concentration and homogeneity and standard guideline criteria were used to determine acceptance cut-offs.
Details on mating procedure:
The number of animals included in the study was the lowest considered necessary to provide an acceptable database (but additional animals were included in the design to cover mating errors or to meet the minimum litter parameter criteria to ensure acceptable foetal evaluations were completed.
98 time mated rats were obtained from the supplier, 96 were allocated to the study and two retained as spares. The animals were in three sub-batches representing progression of gestation (GD 0 defined as day mating was detected) the animals were supplied at GD1, GD2 or GD3.

Since the second sub-batch contained a high proportion of non-pregnant animals, with mating errors suspected (based on foetal weights at necropsy on GD20), an additional group of 32 pregnant rats was included as a replacement group for sub-batch GD2 .
At the time of mating the rats were circa 9 weeks old and in a weight range of 186-324g.

Duration of treatment / exposure:
Oral gavage administration from gestation day 6 to 19 inclusive.
Frequency of treatment:
Daily gavage administration
Duration of test:
animals were terminated on Gestation day 20
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 800 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
32 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
Partial reflux of the dosing formulations on several occasions meant that the precise dose volume administered to certain rats could not be determined. Single animals were affected on GD 6, 7, 8, 13, 14 affecting rats in the control group or group 3 (200 mg/kg bw/day).

The oral route of administration was selected as a possible route of human exposure. Dose levels were selected based on a review of pre-existing data.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:Twice daily checks for clinical signs of reaction to treatment and checks for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 4 and gestation days 6-20

FOOD CONSUMPTION : Yes
- Food consumption measured quantitatively from Day 4 and at daily intervals throughout the remainder of gestation

WATER CONSUMPTION: Yes
- Time schedule for examinations: visual assessment of water bottles at regular intervals

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovarian and uterine eaxminations, macroscopic examination, organ weights for thyroid, parathyroid and liver.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes and distribution
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:placentae - shape, size and colour abnormalities;
Fetal examinations:
- External examinations: Yes: all per litter (late resorptions and foetuses also examined for external abormalities where possible
- Soft tissue examinations: Yes: half per litter - thoracic and abdominal visceral examinations completed
- Skeletal examinations: Yes: half per litter , and extent of ossification determined in each case
- Head examinations: No data
Statistics:
Means and standard deviations were calculated for body weight, food consumption, pregnancy data and organ weights.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Body weight and food consumption data was analysed for homogeneity of variance using the 'F-max' test. If the group variances appeared homogenous, a parametric ANOVA was used an pairwise comparisons were made using Fisher's F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Organ weights were analysed using ANOVA as above and as a percentage of terminal body weight, which was also analysed using ANOVA as an exploratory analysis. In the ANOVA summary tables, the results are reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison. Actual p values are not reported in the summary tables for these analyses.
Fetal weight data was subjected to Kruskal-Wallis non-parametric analysis.
Indices:
No further information
Historical control data:
No information

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no unscheduled deaths among the dams.
There were increases in incidence of ploughing behaviour and excess salivation at 200 and 800 mg/kg bw/day, occurring immediately post-dosing for approximately one hour, and noted between GD 14-18, on a few occasion for each animal. Other signs observed in the high dose group included irregular respiration, subdued behaviour and rolling gait and isolated incidence of vaginal discharge. Other signs were incidental background changes.

There was a slight body weight loss between GD 6 and 7 and reduced weight gain over GD 6-8 for the rats of the high dose group.

Food consumption was lowered in all groups, including the controls, on GD 7. The 200 and 800 mg/kg bw/day groups had consumption decreases of 30-50% and were significantly different from controls. Some individual decreases were as high as 90% in the high dose group but recovery was apparent from GD8. At the low dose level food consumption was comparable to controls throughout the study.

Necropsy of the dams revealed no findings associated with TMPDE-90 treatment.

Increases in absolute and relative liver weights, of between 5 and 15%, were recorded for dams dosed at 200 or 800 mg/kg bw/day. There was no microscopic observation of hypertrophy in the liver. The liver and thyroid weights for rats dosed at 50 mg/kg bw/dya were similar to controls.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Slight food consumption decreases and liver weight changes at 200 or 800 mg/kg bw/day and bodyweight effects in the high dose group.

Details on embryotoxic / teratogenic effects:
Foetal necropsies revealed no macroscopic abnormalities associated with TMPDE-90 treatement. The foetuses from the original sub-batch 2 animals were considered an abnormal size at termination on GD20 and consequently the group was considered to have been mis-mated and replacement animals were included in the study. Similar weight effects were not apparent for foetuses from this replacement set of animals.

The pregnancy performance parameters assessed at GD20 showed no effects of treatment with TMPDE-90. Intergroup variations in numbers of corpora lutea and live/dead implant incidence were considered due to differences in the numbers of litters per group rather than being attributable to TMPDE-90 administration at dose levels of 50 to 800 mg/kg bw/day.

The type and distribution of major and minor foetal abnormaliities and the degree of skeletal ossification showed no changes indicative of a treatment related effect following TMPDE-90 administration to the dams at 50, 200 or 800 mg/kg bw/day. One incidental finding affecting only one foetus in the high dose group was identified as not being within the normal background range of foetal changes - a malpositioned ductus arteriosus, but there was no indication that the finding was treatment related.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

All formulations prepared for use during Weeks 1 and 2 of the original dosing and Week 1 of the additional dosing period were found to be within the acceptance criteria of ±10% of theoretical concentration, with the exception of Group 2 formulations on Week 1 of the additional dosing; these were found to be +11.0% of theoretical concentration.

Following a review of formulation preparation and analysis data the reason for the out of specification results could not be established, and Group 2 back-up samples from this timepoint were analysed using a new standard curve and quality control sample; they were found to be within -1.7% the acceptance criteria. As the original results were only slightly out with the acceptance criteria and the back samples were found to be within specification, it was considered that the Group 2 formulations were suitable for dosing.

Applicant's summary and conclusion

Conclusions:
No evidence of developmental toxicity were observed in this study at dose levels of up to 800 mg/kg bw/d.
Executive summary:

The effects of administration of TMPDE-90 to pregnant rats during the period of organogenesis were studied following oral dosing of groups of 32 rats at doses of 0, 50, 200 or 800 mg/kg bw/d. Time-mated female Sprague-Dawley rats were allocated to three treated groups and one vehicle treated control. The dose volume was 5 ml/kg bw and controls were dosed with corn oil alone.

Rats were treated from Days 6 -19 of gestation (the day mating was detected was defined as Gestation day 0).

The animals were monitored for clinical signs of reaction to treatment, bodyweight changes and food consumption.

The dams were terminated on gestation day 20 and examined for pregnancy and foetal development.

TMPDE-90 doses of 200 or 800 mg/kg bw/d were associated with increased incidence of the behaviour change - ploughing, where the rats lower their nose and plough through cage substrate, excessive salivation following dosing. Group mean food consumption was lower in these two groups and liver weights were also increased in comparison with controls for rats dosed at 200 or 800 mg/kg bw/d. Slight transient bodyweight losses were noted in the high dose group between days 6 -7 and reduced gains over days 6 -8.

No toxicologically significant effects were apparent at the low dose, 50 mg/kg bw/d, which was well tolerated and there were no treatment-related effects apparent in clinical observations, bodyweights, food consumption, organ weight changes or necropsy.

The pregnancy performance and foetal weights were examined at Day 20 and there were no notable differences between treated and control groups. There were no patterns in the response for type and distribution of foetal abnormalities, visceral or skeletal abnormalities or skeletal ossification parameters that indicated any treatment- relationship.

Based on the results of this study the maternal NOEL was 50 mg/kg bw/d, based on slight food consumption decreases and liver weight changes observed at 200 or 800 mg/kg bw/d and bodyweight changes in the high dose group.

The developmental toxicity NOAEL was found to be 800 mg/kg bw/d.