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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-02-24 to 2013-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study on screening guideline.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: Ethyl-3-methyl-3-phenyloxirane-2-carboxylate
- Physical state: clear colourless liquid
- Analytical purity: 99.5 %
- Lot/batch No.: AS00075464
- Expiration date of the lot/batch: 10 May 2013
- Storage condition of test material: room temperature in the dark (neat test item used under safety lighting)

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: Approximately eleven weeks old
- Weight at study initiation: Males weighed 282 to 337g, the females weighed 176 to 224g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding. 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.
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C
Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was available ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
- Acclimation period: The animals were acclimatised for thirteen days during which time their health status was assessed. During
this acclimatisation period preparation of the test site (i.e. clipping) and bandage training
for five consecutive days was performed. On the initial day of bandage training, the
bandages were only applied for approximately 3 hours, for the remaining days bandage
training matched the exposure period applied when animals were being treated.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 50 ± 20% Relative
humidity exceeded this target range on two transient occasions (achieved range 45-
72 %RH) but these deviations were considered to have had no impact on the scientific
validity or integrity of the study.
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled
to give twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:
dermal
Vehicle:
other: 2% Carboxy methylcellulose/1% Tween 80
Details on exposure:
TEST SITE
- Area of exposure: the dorso-lumbar region
- % coverage: approximately 10% of the total body surface area
- Type of wrap if used: The site of application was semioccluded
using a piece of porous gauze covered with a self-adherent bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: The test item formulation removed from the dosing test site and the exposed area was decontaminated with 0.9% saline.
- Time after start of exposure: Following six hours of exposure.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The dose volume employed was 3 ml/kg
- Concentration (if solution): The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted at regular intervals to ensure the dosage for each dosage group was maintained; 0, 33.33, 100, 333.33 mg/mL.
- Constant volume or concentration used: yes
- For solids, paste formed: Not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount(s) applied (volume or weight with unit): The dose volume employed was 3 ml/kg
- Concentration (if solution): No data

USE OF RESTRAINERS FOR PREVENTING INGESTION: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Harlan
Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study. Results show
the formulations to be stable for at least eighteen days. Formulations were therefore
prepared fortnightly and stored at approximately 4ºC in the dark.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: For a maximum of fourteen days. Animals were separated during treatment and whilst in bandages and returned to the mating cages following removal of the bandages.
- 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).
- After successful mating each pregnant female was caged: Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
The test item formulation was
applied daily to the exposed region by a plastic syringe for fifty-one consecutive days for
males, and up to Day 19 of gestation for females.
Frequency of treatment:
Daily
Duration of test:
Fifty-one days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health and behavioural change
immediately before each application, one hour after application and after removal of the
porous gauze and decontamination of the test site. All observations were recorded.
Local Irritation:
Prior to each application of the test item, the dose test site was examined for any signs of
irritation. Any irritation observed was scored according to the scheme described by
Draize J H (1959).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for
males until termination and weekly for females until mating was evident. Body weights
were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and
4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE
During the pre-pairing period, weekly food consumption was recorded for each cage of
adults until pairing. This was continued for males after the mating phase. For females
showing evidence of mating, food consumption was recorded for the periods covering
post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption
was recorded during the lactation period (Days 1-4).
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 CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

Necropsy:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed
by exsanguination on Day 52. Adult females were killed by intravenous overdose of a
suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving
offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any
females which failed to achieve pregnancy or produce a litter were killed on or after Day
25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of
uterine implantations in each horn was recorded. This procedure was enhanced; as
necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski
1964). For pregnant females the number of corpora lutea in the ovaries was also
recorded.
All adult animals and offspring, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free
from fat and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Coagulating gland
Prostate
Epididymides♦
Seminal vesicles
Gross lesions
Testes♦
Ovaries
Treated/untreated skin
Mammary gland (females only)
Uterus/Cervix
Pituitary
Vagina
The tissues from control and 1000 mg/kg bw/day dose group animals and any animals
which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks,
sectioned at a nominal thickness of 5 μm and stained with Haematoxylin and Eosin for
subsequent microscopic examination. In addition, sections of testes and epididymides
from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-
Schiff (PAS) stain and examined. Detailed qualitative examination of the testes was
performed taking into account the tubular stages of spermatogenic cycle.

♦ = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
Fetal examinations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring
was recorded. Offspring were individually identified within each litter by tattoo on Day 1
post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were
calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Necropsy:
All offspring, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.
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 analysed using the decision tree from the ProvantisTM Tables and Statistics
Module as detailed below:
Where appropriate, data transformations were performed using the most suitable
method. The homogeneity of variance from mean values was analysed using Bartlett’s
test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA
with appropriate covariates. Any transformed data were analysed to find the lowest
treatment level that showed a significant effect, using the Williams Test for parametric
data or the Shirley Test for non-parametric data. If no dose response was found, but the
data shows non-homogeneity of means, the data were analysed 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 (nonparametric).
Data not analysed by the Provantis data capture system were assessed separately using
the SPSS statistical package. Initially, the homogeneity of the data was assessed using
Levene’s test.
Indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating
period of the parental generation:
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/ Number of animals mated) x 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and
parturition period of the parental generation:
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating
and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring/ Number of pregnant females) x 100

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were
first calculated for each litter and the group mean was calculated using their individual
litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:
% pre–implantation loss = ((Number of corpora lutea - Number of implantation sites) /Number of corpora lutea) x100

% post–implantation loss = ((Number of implantation sites - Total number of offspring born)/Number of implantation sites) x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) =(Number of offspring alive on Day 1/ Number of offspring born) x 100

Viability Index (%) = (Number of offspring alive on Day 4)/Number of offspring alive on Day 1) x 100

iii) Sex Ratio (% males) was calculated.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths on the study.

Clinical Observations
Clinical signs observed on the study were minimal and were unrelated to treatment.

Body Weight
Body weight gain was unaffected by treatment for both sexes throughout the study,
which included for females, gestation and lactation phases, at 100, 300 and 1000 mg/kg
bw/day.
At 300 and 1000 mg/kg bw/day higher bodyweight gain for males, attained statistical
significance compared to control during Days 15 to 22 but there was no dosage
relationship. These differences were considered to reflect normal biological variation and
were of no toxicological significance.

Food Consumption and Food Efficiency
Food consumption and food conversion efficiency was considered to have been
unaffected by treatment for both sexes throughout the study, which included for females,
gestation and lactation phases, at 100, 300 and 1000 mg/kg bw/day. At 300 and 1000 mg/kg bw/day food intake was higher than control during the final week
of gestation. Although statistical significance was reached at the highest dosage, these
differences were considered to reflect normal biological variation rather than an effect of
treatment. Additionally food consumption during lactation was lower than control at all
dosages during lactation; differences failed to attain statistical significance and there was
no dosage relationship, The lower food consumption was considered to reflect
particularly high food intake for two control females and was unrelated to treatment.

Reproductive Performance
Mating
There was no adverse effect of treatment on mating performance at 100, 300 and
1000 mg/kg bw/day.
It was noted that some treated animals show a longer pre-coital interval than their control
counterparts; the incidence and distribution of these animals did not indicate any dosage
relationship and this finding was considered to be incidental and unrelated to treatment.
For the majority of animals observed evidence of mating was good; one control female
(No. 18) and one at 100 mg/kg day (No. 36) showed poor evidence of mating but only
the control animal failed to achieve pregnancy.

Fertility
There was no adverse effect of treatment on fertility at 100, 300 and 1000 mg/kg bw/day.
At both 300 and 1000 mg/kg bw/day two females failed to achieve pregnancy, however
three of the control females also failed to achieve pregnancy. This slightly lower than
anticipated pregnancy rate may reflect the daily separation of the males and females
during the two week pairing period as part of dermal dose administration procedure.

Gestation Length
No treatment-related effects were detected in the length of gestation between control and
treated groups, with all littering females showing a gestation length between 22 ½ and 24
days.

Necropsy
Adult macroscopic necropsy findings were restricted to small and flaccid testes and small
epididymides for one male at 1000 mg/kg bw/day. This animal failed to induce
pregnancy in its female partner. In isolation this finding was considered to be incidental
and unrelated to treatment.

Organ Weights
Mean absolute and body weight relative testis and epididymis organ weights were
unaffected by treatment at 100, 300 and 1000 mg/kg bw/day.

Histopathology
Examination of the skin (test site) and reproductive organs for both sexes did not indicate
any adverse effect of treatment.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
As previously discussed, three control females and two females at 300 and at 1000
mg/kg bw/day failed to achieve pregnancy; additionally at 1000 mg/kg bw/day, one
pregnant female (implantation sites detected in uterus) was not observed to give birth to
a litter. The following assessment is principally based on the group sizes of 7, 10, 8 and
7 females that successfully reared young to Day 5 of age at 0 (control), 100, 300 and
1000 mg/kg bw/day respectively.

Offspring Litter Size, Sex Ratio and Viability
There was no effect of maternal treatment on the mean number of corpora lutea and
implantations, pre- and post-implantation loss, number of offspring born or subsequent
offspring survival to Day 4 of age, mean litter size or sex ratio on Day 1 or Day 4 of age
at 100, 300 and 1000 mg/kg bw/day.
One female at 1000 mg/kg bw/day was not observed to have given birth to litter although
necropsy revealed two implantation sites in the uterus. It is suspected that this female
may have lost these offspring in utero. It is not unusual for females with such a small
litter to fail to maintain them to parturition and, this finding, in isolation, was considered
incidental and unrelated to treatment.

Offspring Growth and Development
There was no obvious adverse effect of maternal treatment on mean offspring body
weight or litter weight on Day 1 or Day 4 of age at 100, 300 and 1000 mg/kg bw/day.
Surface righting ability of the offspring on Day 1 was also unaffected by treatment at these dosages. Offspring clinical signs were unremarkable and did not indicate any
adverse effect of treatment on offspring development.

Necropsy
Offspring necropsy findings were unremarkable and did not indicate any adverse effect
on offspring development.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dermal administration of Ethyl-3-methyl-3-phenyloxirane-2-carboxylate at dosages up to

1000 mg/kg bw/day was well tolerated by the adult animals with no adverse effects on

survival, clinical condition, body weight gain, food consumption or macroscopic necropsy

findings and subsequent microscopic evaluation. Within the context of this study, the No

Observed Effect Level (NOEL) for adult toxicity was 1000 mg/kg bw/day.

Within the study some treated animals showed a longer pre-coital interval than their control

counterpart. The incidence and distribution of these animals showed no dosage

relationship and was unrelated to treatment. The longer pre-coital interval observed most

probably reflects the separation of the male and female during the dermal dosing

procedure, with some females possibly being separated from their male partner just as

they approached their most receptive point for conception. This may also have resulted

in fertility in the study being slightly lower than anticipated (82.5%); this was clearly

unrelated to treatment as the lowest pregnancy rate occurred in the control group.

The lower pregnancy rate resulted in only seven control female achieving pregnancy and

therefore only seven litters available for assessment. At 1000 mg/kg bw/day, one female

not observed to have given birth to a litter and was therefore suspected as having lost

her litter in utero as necropsy revealed two implantation sites in the uterus. This

occurrence was considered incidental and unrelated to treatment but did result in the

number of litters available for assessment at this dosage also being reduced to seven.

While the number of litters in both groups is slightly lower than ideal (the minimum

generally expected is eight) it is considered that, in this instance, this has had no impact

on the ability of this study to screen for effects on offspring, survival, growth and

development. Results for the study were clear with no equivocal findings observed that

may have benefited from an additional litter being available in either group. The ‘No

Observed Effect Level’ (NOEL) for reproduction and offspring, survival, growth and

development was therefore considered to be 1000 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
Dermal administration of Ethyl-3-methyl-3-phenyloxirane-2-carboxylate at dosages up to
1000 mg/kg bw/day was well tolerated. Within the context of this study the No Observed
Effect Level (NOEL) for adult toxicity and for reproduction and offspring, survival, growth
and development was 1000 mg/kg bw/day.