Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Identification : 2,3-epoxypropyl neodecanoate
Physical State/Appearance : Colorless clear liquid
Purity : 100%
Batch Number : PV7G0064
Label : CARDURA (TM) E-10P, Lot Number: PV7G0064,
Date Received : 02 March 2018
Storage Conditions : Room temperature in the dark
Expiry Date : 02 March 2019

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Animal Information
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 182 to 304 g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, EnvigoRMS(UK) Limited, Oxon, UK) was used. A certificate of analysis of the batch of diet used is given in Annex2. 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 (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the EnvigoResearch Limited, 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 computerizedsystem, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC 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 randomly allocated to treatment groups using a randomization procedure based on stratified bodyweight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Arachis Oil. The stability and homogeneity of the test item formulations were determined by EnvigoResearch Limited, Shardlow, UK Analytical Services. Results are given in Annex 1 and show the formulations to be stable for at least 23 days when stored refrigerated. Formulations were therefore prepared once and stored at approximately +4 °C in the dark.

Samples were taken of each test item formulation and were analyzed for concentration of 2,3-epoxypropyl neodecanoate at Envigo Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in Annex 1. The results indicate that the prepared formulations were within1% of the nominal concentration.

Animals were allocated to treatment groups as follows:

Treatment Dose Level Treatment Concentration Animal Numbers
Group (mg/kg bw/day) Volume (mL/kg) (mg/mL)
Control 0 4 0 24 (1-24)
Low 100 4 25 24 (25-48)
Intermediate 300 4 75 24 (49-72)
High 1000 4 250 24 (73-96)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of 2,3-epoxypropyl neodecanoate at Envigo Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in Annex 1. The results indicate that the prepared formulations were within 1% of the nominal concentration.
Duration of treatment / exposure:
The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Frequency of treatment:
Once per day
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
The study was performed to investigate the effects of the test item on embryonic and fetal development, following repeated administration by gavage atdose levels 100, 300 and 1000 mg/kg bw/day to the Sprague-Dawley Crl:CD® (SD) IGS BR strain rat during gestation, including the period of organogenesis.

The dose levels were selected in collaboration with the Sponsor Representative and were based on available toxicity data including preliminary data from a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Envigo Study Number: BT01NH). In the preliminary study, a dose level of 1000 mg/kg bw/day caused initial reductions in body weight gains and food consumptions. However, these effects were considered not to be sufficient enough to preclude the use of this as the high dose level for further investigation.

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

The study was performed between 12 June 2018 and 03 September 2018. The in-life phase of the study was conducted between 15 June 2018 (first day of treatment) and 04 July 2018 (final day of necropsy).

Examinations

Maternal examinations:
General Observations/Measurements
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Bodyweights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Ovaries and uterine content:
Terminal Investigation
Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboardtags marked with chinagraph pencil andplacedinto70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin redSbefore being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Fetal examinations:
Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:

(number of corpora lutea - number of implantations) x 100/number of corpora lutea

Percentage post-implantation loss was calculated as:

(number of implantations - number of live fetuses) x 100/number of implantations

Sex Ratio
Sex ratio was calculated as:

% male fetuses (sex ratio) = (Number of male fetuses x 100)/Total number of fetuses
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:

Female body weight, female body weight change, food consumption, gravid uterus weight, all caesarean necropsy parameters and fetal parameters, fetal evaluation parameters, including skeletal or visceral findings.

Data was assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett's test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett's test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal- Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression.

Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations of toxicological significance at any dose level.

Sporadic incidences of increased salivation were evident in the majority of animals treated with 1000 mg/kg bw/day from Day 5 until Day 19 of gestation. Isolated instances of increased salivation were noted in seven animals treated with 300 mg/kg bw/day from Day 8 until Day 17 of gestation but to a much lesser extent than animals treated with 1000 mg/kg bw/day. Such findings are commonly observed in this type of study and usually reflect unpalatability and/or an irritant nature of the test item formulation and are generally deemed to be of no toxicological importance.

No clinical signs were apparent in any animal treated with 100 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Description (incidence and severity):
Animals treated with 300 and 1000 mg/kg bw/day showed marginal reductions in body weight gains during the majority of the treatment period with a dose relationship being apparent, however, statistical significance was not achieved. The reductions in body weight gains throughout the treatment period ultimately resulted in cumulative body weight gains being statistically significantly reduced (p<0.05) from Day 14 to Day 17 in animals treated with 300 mg/kg bw/day and from Day 8 until the end of the treatment period at 1000 mg/kg bw/day (p<0.05-p<0.01). This resulted in overall body weight gains for these animals being 9% and 12% lower than control for animals treated with 300 and 1000 mg/kg bw/day respectively. When adjustment was made for the gravid uterus, statistically significant reductions of 20% (p<0.05) and 36% (p<0.001) were noted in animals treated with 300 and 1000 mg/kg bw/day respectively.

No effects were detected on body weight development in females treated with 100 mg/kg bw/day.
Description (incidence and severity):
Animals treated with 1000 mg/kg bw/day showed reductions in food consumption throughout the treatment period which achieved statistical significance from Days 3 to 5, 5 to 8, 11 to 14 and 17 to 20 (p<0.05 to p<0.001).

Animals treated with 300 mg/kg bw/day showed reductions in food consumption throughout the treatment period which achieved statistical significance from Day 5 until the end of the treatment period (p<0.05 to p<0.001).

No effect was detected on food consumption in females treated with 100 mg/kg bw/day.
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Details on results:
No toxicologically significant macroscopic abnormalities were detected at necropsy.

One animal treated with 300 mg/kg bw/day exhibited generalized fur loss, however, due to the isolated nature of this finding it was considered not to be related to treatment with the test item.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no effects

Results (fetuses)

Description (incidence and severity):
The following assessment is based on the 23, 23, 23 and 20 females with live young on Day 20 of gestation at 0 (control), 100, 300 and 1000 mg/kg bw/day, respectively.

There was no effect of maternal treatment on litter data as assessed by the mean number of implantations, in-utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size and post-implantation losses at 100, 300 or 1000 mg/kg bw/day.

Pre-implantation losses and sex ratios across all test item-treated dose groups were also similar to controls.

There was no obvious effect of maternal treatment on litters as assessed by litter, fetal or placental weights.

For litters from dams treated with 300 and 1000 mg/kg bw/day, mean male and/or female fetal and combined fetal weights were statistically significantly lower (p<0.05 - p<0.01 and p<0.05 respectively) than control. However, the differences at each dosage were slight and an association with treatment is considered equivocal, especially as a true dose relationship was not apparent. The slightly lower fetal weights were considered to be a reflection of the slightly larger litter sizes apparent at these dosages when compared to control and as such, this apparent effect was consideredto be due to normal biological variation. Placental weights, total placental weights and litter weights were similar to control.

There was no effect of maternal treatment at 100 mg/kg bw/day on litters as assessed by litter, fetal or placental weights.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Neither the type, incidence nor distribution of external findings apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

At 1000 mg/kg bw/day statistically significant increases in the incidence of increased renal pelvic cavitation (p<0.05) was noted. The percentage incidence was found to be within the historical control data ranges. As a dose-related response was not evident, this finding is considered to be due to normal biological variation and therefore, un-related to treatment with the test item.

At 100 mg/kg bw/day statistically significant increases in the incidences of kinked ureter (p<0.01), increased renal pelvic cavitation (p<0.001) and absence of the renal papilla (p<0.05) were noted. All findings were found to be higher than historical control data ranges. However, as a dose relationship was not apparent, these findings are considered to be incidental and un-related to treatment with the test item.

Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

At 1000 mg/kg bw/day a statistically significant increase (p<0.05) in the incidence of fetuses/litters exhibiting incomplete ossification (nasal) was apparent. A higher percentage of each litter showing this effect was noted in seven litters at this dose level when compared with the historical control data range, however, the overall percentage of litters affected was within the historical control data range. There was no true dose related response and this variation was seen in the absence of other delays in ossification and any adverse effects on mean fetal weight. This was considered to be an isolated finding which was not regarded as evidence of an effect of embryofetal developmental toxicity.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

KEY TO TABLES AND APPENDICES

M       Male

F       Female

sd       Standard deviation

n       Number of animals/litters

NF       Number of fetuses

NL       Number of litters

%†       Group mean percent

N/A       Not applicable

NP       Not pregnant

V       Viable fetuses+

FWT       Fetal weight

PWT       Placental weight

C       Cervix

DF       Dead fetus

ED       Early death

LD       Late death

      

Statistical Footnotes:

*       Significantly different from control group p<0.05

**       Significantly different from control group p<0.01

***       Significantly different from control group p<0.001

TABLES

Table 1       Summary of Female Performance

Category       Number of Females at Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

Initial Group Size       24       24       24       24

Pregnant       23       23       23       20

Non-Pregnant       1       1       1       4

Table 2       Summary Incidence of Daily Clinical Observations

Dose Level (mg/kg bw/day)       Number of Animals       Clinical Observations       Number Showing Effect

(Days post coitum affected)

0 (Control)       24       No abnormalities detected       

100       24       No abnormalities detected       

300       24       Increased salivation       7 (8, 14, 15, 17)

1000       24       Increased salivation       23 (5, 6, 8-19)

Table 3       Group Mean Body Weight Values

Dose Level (mg/kg bw/day)              Body Weight (g) on Day of Gestation

             3       4       5       8       11       14       17       20

0 (Control)       mean       266.0       270.7       276.0       291.1       309.1       327.2       358.6       403.4

      sd       30.7       33.0       31.8       32.7       33.8       34.7       37.6       42.1

      n       23       23       23       23       23       23       23       23

100       mean       268.0       272.2       276.4       291.7       309.2       327.9       358.9       402.8

      sd       23.4       24.4       23.9       24.1       24.4       27.6       27.8       34.0

      n       23       23       23       23       23       23       23       23

300       mean       263.8       266.9       271.0       284.3       301.1       316.0       345.6       388.7

      sd       25.6       25.7       25.0       24.0       23.6       22.2       22.8       26.4

      n       23       23       23       23       23       23       23       23

1000       mean       262.4       264.6       267.4       281.5       298.4       315.4       343.3       382.8

      sd       26.4       29.7       27.0       26.4       27.6       27.3       28.8       33.2

      n       20       20       20       20       20       20       20       20

Table 4       Group Mean Body Weight Change Values

Dose Level (mg/kg bw/day)              Body Weight Change (g) during Days of Gestation

             3 to 4       4 to 5       5 to 8       8 to 11       11 to 14       14 to 17       17 to 20

0 (Control)       mean       4.7       5.3       15.0       18.0       18.1       31.3       44.8

      sd       4.0       5.3       4.5       5.0       4.7       5.7       9.2

      n       23       23       23       23       23       23       23

100       mean       4.2       4.2       15.3       17.5       18.7       31.0       43.9

      sd       5.9       4.8       5.6       3.2       5.0       5.9       8.0

      n       23       23       23       23       23       23       23

300       mean       3.1       4.1       13.3       16.8       14.8       29.7       43.1

      sd       4.5       4.8       6.0       3.6       4.7       5.3       7.2

      n       23       23       23       23       23       23       23

1000       mean       2.2       2.8       14.1       17.0       17.0       27.9       39.6

      sd       5.8       6.1       5.0       3.8       4.8       5.7       12.9

      n       20       20       20       20       20       20       20

Dose Level (mg/kg bw/day)              Cumulative Body Weight Change (g) from Day 3 of Gestation

             4       5       8       11       14       17       20

0 (Control)       mean       4.7       10.0       25.0       43.0       61.2       92.5       137.3

      sd       4.0       5.1       6.8       9.4       12.3       16.2       23.4

      n       23       23       23       23       23       23       23

100       mean       4.2       8.4       23.7       41.2       59.9       90.9       134.8

      sd       5.9       7.6       8.0       8.5       11.3       11.7       17.0

      n       23       23       23       23       23       23       23

300       mean       3.1       7.2       20.6       37.3       52.2*       81.8*       124.9

      sd       4.5       4.7       6.5       6.9       9.6       12.1       14.6

      n       23       23       23       23       23       23       23

1000       mean       2.2       5.0       19.1*       36.0*       53.0*       80.9*       120.4**

      sd       5.8       6.5       6.8       7.2       10.0       9.9       17.2

      n       20       20       20       20       20       20       20

Table 5       Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

Dose Level (mg/kg bw/day)              Body Weight (g) on Days of Gestation       Body Weight Change (g) during Days of Gestation       Gravid Uterus Weight

(g)       Adjusted

Body Weight (g)

Day 20       Adjusted

Body Weight Change (g)

3-20

             3       20       3-20                     

0 (Control)       mean       266.0       403.4       137.3       83.517       319.9       53.8

      sd       30.7       42.1       23.4       18.520       36.8       13.8

      n       23       23       23       23       23       23

100       mean       268.0       402.8       134.8       83.682       319.1       51.1

      sd       23.4       34.0       17.0       16.383       29.8       15.4

      n       23       23       23       23       23       23

300       mean       263.8       388.7       124.9       82.086       306.6       42.8*

      sd       25.6       26.4       14.6       9.296       23.5       11.3

      n       23       23       23       23       23       23

1000       mean       262.4       382.8       120.4*       85.787       297.0       34.6***

      sd       26.4       33.2       17.2       9.915       34.1       18.9

      n       20       20       20       20       20       20

Table 6       Group Mean Food Consumption Values

Dose Level (mg/kg bw/day)              Food Consumption (g/rat/day) between Days of Gestation

             3 - 5       5 - 8       8 - 11       11 - 14       14 - 17       17 - 20

0 (Control)       mean       21.4       23.2       25.4       25.3       25.8       27.1

      sd       3.0       2.8       2.6       2.9       3.3       3.1

      n       23       23       23       23       23       23

100       mean       21.4       22.3       24.8       24.8       25.5       26.5

      sd       3.1       2.1       2.5       3.1       2.7       3.2

      n       23       23       23       23       23       23

300       mean       20.0       20.5***       23.2**       22.3***       23.5*       25.0*

      sd       1.9       1.8       2.0       2.0       2.1       1.9

      n       23       23       23       23       23       23

1000       mean       18.9*       20.1***       23.7       23.2*       24.3       24.7*

      sd       4.1       2.5       2.5       1.8       2.1       2.9

      n       20       20       20       20       20       20

Table 7       Summary Incidence of Necropsy Findings

      Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

TERMINAL DEATH                            

                           

Number of animals examined       24       24       24       24

External:                            

Generalized fur loss       0       0       1       0

No abnormalities detected       24       24       23       24

Table 8       Group Mean Litter Data Values

Dose Level (mg/kg bw/day)              Number of Corpora Lutea       Number of Implants       Number of Embryonic/Fetal Deaths       Implantation Loss

%       Number of Live Implants       %

Male Fetuses       Mean Male Fetal Weight (g)       Mean Female Fetal Weight (g)       Mean Fetal Weight (g)       Mean Placental Weight

(g)       Litter Weight (g)       Total Placental Weight

(g)

                           Early       Late       Total       Pre       Post       Male       Female       Total                                                 

0 (Control)       mean       15.9       13.7       0.3       0.0       0.4       14.7       2.6       6.5       6.8       13.3       50.8       4.150       3.915       4.046       0.550       53.385       7.181

      sd       3.3       3.2       0.6       0.2       0.8       14.7       4.9       2.1       2.4       3.1       15.3       0.300       0.278       0.295       0.055       12.723       1.571

      n       23       23       23       23       23       23       23       23       23       23       23       23       22       23       23       23       23

100       mean       16.2       14.5       0.7       0.1       0.7       10.7       5.8       6.9       6.8       13.7       50.6       3.998       3.784       3.876       0.539       53.276       7.334

      sd       2.2       2.4       1.2       0.3       1.2       10.6       10.8       3.0       3.1       3.0       18.8       0.248       0.216       0.218       0.045       11.514       1.452

      n       23       23       23       23       23       23       23       23       23       23       23       23       23       23       23       23       23

300       mean       16.0       14.1       0.3       0.1       0.3       11.7       2.5       6.9       6.9       13.8       50.3       3.915*       3.707*       3.810**       0.530       52.317       7.261

      sd       1.8       1.9       0.5       0.3       0.6       10.2       4.6       2.2       2.4       2.0       14.9       0.270       0.217       0.230       0.050       6.988       1.010

      n       23       23       23       23       23       23       23       23       23       23       23       23       23       23       23       23       23

1000       mean       16.5       14.9       0.4       0.3       0.7       9.4       4.3       6.6       7.6       14.2       46.1       3.967       3.709*       3.827*       0.570       54.337       7.969

      sd       2.1       1.9       0.5       0.4       0.7       7.1       5.0       2.1       1.8       1.9       11.3       0.321       0.300       0.302       0.149       8.218       1.649

      n       20       20       20       20       20       20       20       20       20       20       20       20       20       20       20       20       20

Table 9       Summary Incidence of Fetal External Findings

External Findings       Dose level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of fetuses (litters) examined

      305 (23)       316 (23)       317 (23)       284 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

Total Number Affected       8       5       2.5       6       5       2.2       14       10       4.3       21       6       8.3

Hematoma - Right Side of Face       1       1       0.3       1       1       0.3       0       0       0.0       0       0       0.0

Small Fetus       4       2       1.4       4       3       1.6       9       8       2.9       6       4       2.4

Large Placenta       0       0       0.0       1       1       0.3       1       1       0.3       12       2       5.0

Hematoma - Left Hind Limb       0       0       0.0       0       0       0.0       0       0       0.0       1       1       0.4

Small Placenta       2       2       0.6       0       0       0.0       7       6       2.1       4       2       1.4

Four Hind Limbs       1       1       0.3       0       0       0.0       0       0       0.0       0       0       0.0

Hematoma - Right Ear       1       1       0.3       0       0       0.0       0       0       0.0       0       0       0.0

Table 10       Summary Incidence of Fetal Visceral Findings

Visceral Findings       Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of Fetuses (litters) Examined

      157 (23)       165 (23)       164 (23)       147 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

EXTERNAL                                                                                    

Hemorrhage       0       0       0.0       1       1       0.5       0       0       0.0       0       0       0.0

HEAD                                                                                    

Tongue - short       0       0       0.0       0       0       0.0       0       0       0.0       1       1       0.6

Rugae - non-uniform patterning       10       7       6.3       7       5       3.8       13       6       7.7       7       6       4.5

Micropthalmia       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Brain - olfactory ventricle - enlarged       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Brain - lateral ventricle - enlarged       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Brain - third ventricle - enlarged       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Brain - fourth ventricle - enlarged       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

ABDOMEN                                                                                    

Liver - additional lobe between right and left median       0       0       0.0       1       1       0.5       0       0       0.0       5       3       3.2

Liver - lobes thickened throughout       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Liver - papillary process - reduced in size       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Spleen - reduced in size       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Umbilical artery - left-sided       0       0       0.0       0       0       0.0       0       0       0.0       2       2       1.4

Testis - partially undescended       1       1       0.6       1       1       0.5       0       0       0.0       0       0       0.0

Ureter - kinked       4       3       2.6       26       11       15.0**       5       4       3.4       11       8       7.5

Ureter - dilated - Slight/Severe       3       2       2.1       11       7       6.9       3       2       2.2       5       4       3.6

Kidney - malpositioned - Cranially/Caudally       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Kidney - reduced in size       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Renal pelvic cavitation - increased - Slight/Severe       1       1       0.7       23       12       14.5***       7       6       4.2       8       6       5.5*

Table 10 (continued)       Summary Incidence of Fetal Visceral Findings

Visceral Findings       Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of Fetuses (litters) Examined

      157 (23)       165 (23)       164 (23)       147 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

ABDOMEN (continued)                                                                                    

Renal papilla - absent       0       0       0.0       5       4       3.8*       0       0       0.0       1       1       0.6

THORAX                                                                                    

Thymus - lobe partially undescended       5       5       3.3       5       3       3.0       6       5       3.8       3       3       2.0

Thymus - lobe - irregular surface       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Lungs - irregular surface throughout       1       1       0.6       0       0       0.0       0       0       0.0       0       0       0.0

Atrium - enlarged       0       0       0.0       0       0       0.0       0       0       0.0       1       1       0.6

Total       19       14       12.2       41       16       25.6       28       13       17.2       28       12       18.2

Table 11       Summary Incidence of Fetal Skeletal Findings

Skeletal Findings       Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of Fetuses (litters) Examined

      148 (23)       151 (23)       153 (23)       137 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

SKULL                                                                                    

Fontanelle (anterior) - large       0       0       0.0       1       1       2.2       1       1       0.6       2       2       1.3

Nasal - incomplete ossification       6       3       3.7       21       9       13.0       10       8       6.7       25       9       19.0*

Frontal - incomplete ossification       2       2       1.2       4       4       2.8       0       0       0.0       3       3       2.1

Frontal - unossified area       1       1       0.6       3       2       2.0       1       1       0.9       2       2       1.7

Parietal - incomplete ossification       10       8       6.3       9       6       5.9       8       6       5.1       9       5       7.0

Parietal - unossified area(s)       1       1       0.6       1       1       0.5       0       0       0.0       0       0       0.0

Interparietal - incomplete ossification       26       13       17.0       38       13       24.4       37       14       23.9       43       16       32.3

Interparietal - unossified area(s)       1       1       0.5       0       0       0.0       0       0       0.0       1       1       1.0

Occipital (Supra-occipital) - incomplete ossification       17       10       10.7       21       13       14.9       20       11       13.1       25       12       20.0

Occipital (Supra-occipital) - unossified area(s)       17       13       11.1       18       13       15.3       10       8       6.6       7       5       6.1

Squamosal - incomplete ossification       19       9       12.3       20       13       12.8       15       11       9.2       23       9       16.8

Squamosal - unossified area(s)       1       1       0.6       1       1       0.5       0       0       0.0       0       0       0.0

Jugal - incomplete ossification       5       3       3.5       5       3       3.2       6       5       4.4       5       3       3.8

Table 11 (continued)       Summary Incidence of Fetal Skeletal Findings

Skeletal Findings       Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of Fetuses (litters) Examined

      148 (23)       151 (23)       153 (23)       137 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

SKULL (continued)                                                                                    

Zygomatic process of maxilla - incomplete ossification       11       5       7.5       14       9       8.1       15       7       9.7       14       8       9.1

Zygomatic process of squamosal - incomplete ossification       0       0       0.0       1       1       0.7       1       1       0.9       1       1       0.6

Zygomatic process of squamosal - fused to jugal       0       0       0.0       0       0       0.0       1       1       0.6       0       0       0.0

Hyoid - incomplete ossification       16       8       10.9       24       14       14.8       17       10       11.9       22       11       16.8

Hyoid - not ossified       5       4       2.9       12       7       6.7       6       3       3.7       6       5       4.1

Presphenoid - incomplete ossification       1       1       0.6       0       0       0.0       1       1       0.6       2       2       1.3

Basisphenoid - incomplete ossification       0       0       0.0       0       0       0.0       0       0       0.0       1       1       0.7

Basioccipital - misshapen       0       0       0.0       0       0       0.0       0       0       0.0       1       1       1.0

VERTEBRAL COLOUMN                                                                                    

Odontoid - ossification present       2       2       1.6       0       0       0.0       1       1       0.6       0       0       0.0

Ventral arch of vertebra 1 - ossification present       47       17       34.3       50       13       31.3       39       16       27.1       42       12       30.7

Cervical (neural) arch - incomplete ossification       3       2       1.8       7       7       6.2       10       6       6.5       6       4       4.7

Thoracic centrum - incomplete ossification       5       5       3.0       4       4       4.1       15       5       8.9       10       6       7.7

Thoracic centrum - not ossified       0       0       0.0       0       0       0.0       2       2       1.2       2       1       1.4

Thoracic centrum - bipartite ossification       1       1       0.5       0       0       0.0       2       1       1.2       2       2       1.3

Thoracic centrum - dumb-bell-shaped       10       6       6.3       20       8       15.4       17       11       11.2       17       10       12.5

Table 11 (continued)       Summary Incidence of Fetal Skeletal Findings

Skeletal Findings       Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of Fetuses (litters) Examined

      148 (23)       151 (23)       153 (23)       137 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

VERTEBRAL COLOUMN                                                                                    

Thoracic centrum - asymmetrically ossified       1       1       0.5       0       0       0.0       2       2       1.5       1       1       0.6

Lumbar centrum - bipartite ossification       0       0       0.0       0       0       0.0       1       1       0.6       1       1       0.7

Lumbar centrum - dumb-bell-shaped       0       0       0.0       0       0       0.0       1       1       0.6       0       0       0.0

Sacral (neural) arch - incomplete ossification       20       9       12.7       28       13       17.3       28       14       18.5       27       13       21.8

Sacral (neural) arch - not ossified       0       0       0.0       0       0       0.0       0       0       0.0       1       1       1.0

Caudal vertebrae - less than 4 ossified       23       13       14.3       34       15       24.8       39       16       24.4       45       15       33.6

Number of pre-sacral vertebrae = 25/27 (add “D” for pelvic displacement, or comment)       4       4       2.4       0       0       0.0       1       1       0.6       1       1       0.7

RIBS                                                                                    

Ossification centre - associated with 7th cervical vertebra       0       0       0.0       0       0       0.0       0       0       0.0       1       1       0.8

14th rib - extra - associated with 1st lumbar vertebra       1       1       0.5       1       1       0.9       0       0       0.0       0       0       0.0

Ossification centre - associated with 1st lumbar vertebra       16       11       11.0       7       5       4.8       7       6       5.0       13       7       9.0

One or more ribs - thickened       0       0       0.0       0       0       0.0       0       0       0.0       2       2       1.8

Rib - short       2       1       1.1       0       0       0.0       1       1       0.6       0       0       0.0

Rib - rudimentary       0       0       0.0       1       1       0.5       1       1       0.7       1       1       1.0

Rib - fused       0       0       0.0       0       0       0.0       0       0       0.0       1       1       0.7

Rib - not ossified       1       1       0.5       0       0       0.0       0       0       0.0       0       0       0.0

Table 11 (continued)       Summary Incidence of Fetal Skeletal Findings

Skeletal Findings       Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of Fetuses (litters) Examined

      148 (23)       151 (23)       153 (23)       137 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

RIBS (continued)                                                                                    

Costal cartilage - misaligned       5       4       2.9       3       3       3.5       6       4       4.5       3       3       2.3

Costal cartilage - not fused to sternebra       9       7       6.3       13       9       9.9       14       10       9.0       7       5       5.0

STERENBRAE                                                                                    

Sternebra - incomplete ossification       2       1       1.1       4       4       2.9       4       3       2.4       3       3       2.3

Sternebra - not ossified       0       0       0.0       0       0       0.0       1       1       0.7       0       0       0.0

Sternebra - bipartite ossification       1       1       0.5       1       1       2.2       1       1       0.7       0       0       0.0

Sternebra - misaligned       7       5       4.4       5       5       3.8       12       10       8.4       3       3       2.3

Sternebra - fused       0       0       0.0       1       1       0.7       0       0       0.0       0       0       0.0

Sternebra - misshapen       1       1       0.5       0       0       0.0       0       0       0.0       0       0       0.0

Xiphoid cartilage - partially split       8       6       4.4       8       8       5.2       3       3       1.9       6       6       4.2

PECTORAL GIRDLE                                                                                    

Scapula - bent       0       0       0.0       0       0       0.0       0       0       0.0       1       1       1.0

Scapula - misshapen (comment on region)       2       2       1.1       5       4       3.1       2       2       1.2       4       3       2.7

FORELIMBS                                                                                    

Metacarpal - not ossified       37       14       23.0       51       16       33.3       64       19       41.6       64       17       47.4

Metacarpal - incomplete ossification       1       1       0.6       1       1       0.6       1       1       0.7       0       0       0.0

Forepaw phalanges - 1 or more - ossified       28       11       19.3       18       11       12.4       10       6       6.6       15       6       11.6

Humerus - incomplete ossification       1       1       0.6       6       4       5.0       1       1       0.6       5       3       3.7

Table 11 (continued)       Summary Incidence of Fetal Skeletal Findings

Skeletal Findings       Dose Level (mg/kg bw/day)

      0 (Control)       100       300       1000

      Number of Fetuses (litters) Examined

      148 (23)       151 (23)       153 (23)       137 (20)

      NF       NL       %†       NF       NL       %†       NF       NL       %†       NF       NL       %†

FORELIMBS (continued)                                                                                    

Polymelia - forelimb (State details)       1       1       0.5       0       0       0.0       0       0       0.0       0       0       0.0

PELVIC GIRDLE                                                                                    

Ischium - incomplete ossification       0       0       0.0       2       2       1.1       1       1       0.6       0       0       0.0

Pubis - not ossified       0       0       0.0       3       2       2.3       0       0       0.0       0       0       0.0

Pubis - incomplete ossification       3       2       2.1       11       7       7.0       3       2       2.1       7       5       5.3

HINDLIMBS                                                                                    

Metatarsal - 1st - ossified       0       0       0.0       1       1       0.7       0       0       0.0       3       1       2.5

Metatarsal - incomplete ossification       0       0       0.0       0       0       0.0       0       0       0.0       3       2       2.6

Femur - incomplete ossification       9       7       9.8       14       10       12.3       6       6       4.2       10       5       8.3

Polymelia - hindlimb (State details)       1       1       0.5       0       0       0.0       0       0       0.0       0       0       0.0

Total       131       23       88.5       135       23       89.9       133       23       87.7       128       20       93.9

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of 2,3-epoxypropyl neodecanoate to pregnant rats from gestation Days 3 to 19, at dose levels of 100, 300 or 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an effect on food consumption at 300 and 1000 mg/kg bw/day. No similar effects were apparent at 100 mg/kg bw/day. Consequently, 100 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL) for the pregnant female.

In-utero survival of the developing conceptus was unaffected by maternal treatment with 1000 mg/kg bw/day There were also no changes in the measured fetal parameters or embryofetal development. The `No Observed Effect Level' (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

 US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

 Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 Nousan No 8147, (24 November 2000)

 OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

 Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (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 twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no clinical observations of any toxicological significance at any dose level.

Body Weight

Animals treated with 300 and 1000 mg/kg bw/day showed marginal reductions in body weight gains during the majority of the treatment period with a dose relationship being apparent. This resulted in reductions in cumulative body weight gains from Day 14 to Day 17 in animals treated with 300 mg/kg bw/day and from Day 8 until the end of the treatment period at 1000 mg/kg bw/day. This resulted in lower overall body weight gains for these animals when compared to control animals. When adjustment was made for the gravid uterus these reductions were more pronounced.

No effects were detected on body weight development in females treated with 100 mg/kg bw/day.

Food Consumption

Animals treated with 300 and 1000 mg/kg bw/day showed general reductions in food consumption throughout the treatment period.

No effect was detected on food consumption in females treated with 100 mg/kg bw/day.

Water Consumption

No effect on water consumption was detected.

Post Mortem Studies

No toxicologically significant macroscopic abnormalities were detected at necropsy.

Litter Data and Litter Placental and Fetal Weights

There was no effect of maternal treatment on litter data as assessed by the mean number of implantations, in-utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size and post-implantation losses at 100, 300 or 1000 mg/kg bw/day. Pre-implantation losses and sex ratios across all test item-treated dose groups were also similar to controls.

There was no obvious effect of maternal treatment on litters as assessed by litter, fetal or placental weights.

Fetal Examination

Skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg/day.

Conclusion

The oral (gavage) administration of 2,3-epoxypropyl neodecanoate to pregnant rats from gestation Days 3 to 19, at dose levels of 100, 300 or 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an effect on food consumption at 300 and 1000 mg/kg bw/day. No similar effects were apparent at 100 mg/kg bw/day. Consequently, 100 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL) for the pregnant female.

In-utero survival of the developing conceptus was unaffected by maternal treatment with 1000 mg/kg bw/day. There were also no changes in the measured fetal parameters or embryofetal development. The `No Observed Effect Level' (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.