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Toxicological information

Toxicity to reproduction

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Administrative data

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

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 421 (Reproduction / Developmental Toxicity Screening Test)
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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxfordshire, United Kingdom
- Age at study initiation: Approximately 11 weeks old
- Weight at study initiation: Males 282 g to 337 g; females 176 g to 224 g
- 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: 22 ºC ± 3 ºC
- 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: ≥ 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

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.
- Time intervals for shavings or clippings:

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
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.
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 4 ºC in the dark.
Duration of treatment / exposure:
The test item formulation was applied daily to the exposed region by a plastic syringe for 51 consecutive days for males, and up to Day 19 of gestation for females.
Frequency of treatment:
Daily
Details on study schedule:
- Groups of 10 male and 10 female animals were dosed according to dose group for 14 days prior to pairing.
- On Day 15, following decontamination of the exposure site, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
- Animals were returned to their original holding cages during the exposure period.
- Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
- Treatment was continued for males until study termination. Pregnant females were treated up to and including Day 19 of gestation.
- Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
- The male dose groups were killed and examined macroscopically on Day 52.
- The termination of the males was delayed (and dosing extended) to allow for the results of mating for the majority of the females to be known prior to male necropsy.
- At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS
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
- 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.
Litter observations:
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 these data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
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: Coagulating gland (males only), prostate (males only), seminal vesicles (males only), gross lesions, ovaries (females only), treated/untreated skin, mammary gland (females only), uterus/cervix (females only), pituitary, Vagina (females only).

Samples of the following tissues were preserved in Bouin’s fluid then transferred to 70 % Industrial Methylated Spirits (IMS) approximately 48 hrs later: Testes (males only), epididymides (males only).

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.
Postmortem examinations (offspring):
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 Provantis 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.
Reproductive 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
Offspring viability indices:
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) x 100

% 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)
Sex ratio was calculated for each litter on Days 1 and 4 post partum, using the following formula: (Number of male offspring/ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.

REPRODUCTIVE PERFORMANCE: 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 22½ 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 (P0)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

As previously discussed, 3 control females and 2 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.

Overall reproductive toxicity

Reproductive 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.