Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Toxicity to reproduction

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:

The study was performed between 17 December 2009 and 19 July 2010. The in-life phase of the study was conducted between 22 December 2009 (first day of treatment) and 14 February 2010 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/2009

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

EC Number:

935-853-6

IUPAC Name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Constituent 2

Reference substance name:

Reaction Mass of Bis(epoxyethyl) benzene and (Ethylphenyl) Oxirane

IUPAC Name:

Reaction Mass of Bis(epoxyethyl) benzene and (Ethylphenyl) Oxirane

Details on test material:

- Name of test material (as cited in study report): Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane
- Substance type: Oragnic, Multiconstituent
- Physical state: clear extremely pale yellow liquid
- Lot/batch No.: 200901317-17
- Date received: 26 May 2009
- Storage condition of test material : approximately 4°C in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan UK, Ltd, Oxon, UK

- Age at study initiation:
Approximately 12 weeks old

- Weight at study initiation:
295 to 348g (male); 195 to 227g (female)

- Fasting period before study:
Not applicable

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). During the mating phase animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. 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:
The animals were allowed free access to food. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.

- Water:
The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.

- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS

- Temperature: (°C): 21 ± 2

- Humidity (%): 55 ± 15

- Air changes (per hr): At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light): 12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
Up to 54 consecutive days for the (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 50, 100 and 200 mg/kg/day.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 0044-1419). Results from the previous study showed the formulations to be stable for at least twenty one days. Formulations were therefore prepared fortnightly during the treatment period and stored at 4ºC in the dark.
Samples of each test material formulation were taken and analysed for concentration of Reaction mass of bis(epoxyethyl) benzene and(ethylphenyl) oxirane at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The results indicate that the prepared formulations were within 10% of the nominal concentration.


DIET PREPARATION
A pelleted diet Rodent 2018C Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study period.

- Rate of preparation of diet (frequency):
Daily

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
No data

VEHICLE
Arachis oil

- Justification for use and choice of vehicle (if other than water):
Not reported

- Concentration in vehicle:
50, 25, 12.5 and 0 mg/ml

- Amount of vehicle (if gavage):
4 ml/kg

- Lot/batch no. (if required):
Not applicable

- Purity:
Not applicable

Details on mating procedure:

- M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)

- Length of cohabitation:
Up to 14 days

- 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 the oestrous cycle 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) and the males were subsequently returned to their original holding cages (unless required for additional pairing).

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
No data

- Further matings after two unsuccessful attempts:
No data

- After successful mating each pregnant female was caged:
Mated females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test material formulations were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/ml. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.

The standard and sample solutions were analysed by HPLC using the following conditions:

HPLC : Agilent Technologies 1200, incorporating autosampler and workstation
Column : Phenosphere NEXT phenyl (250 x 4.6 mm id)
Column temp : 40°C
Mobile phase : Acetonitrile:water (70:30 v/v)
Flow-rate : 1 ml/min
UV detector wavelength : 230 nm
Injection volume : 25 µl
Retention time : ~ 3.9 and 4.5 mins

Duration of treatment / exposure:

Up to Fifty-four consecutive days

Frequency of treatment:

Daily

Details on study schedule:

Chronological Sequence of Study
Dose Groups 1 to 4

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iv) 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.

v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.

ix) Blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day (0 mg/ml)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day (12.5 mg/ml)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 mg/kg/day (50 mg/ml)
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
200 mg/kg/day (100 mg/ml)
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose (including control).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Cros-Reference Study :( Seven Day Repeat Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat Harlan Laboratory Ltd Study Report No. 0044-1419)

- Rationale for animal assignment (if not random):
Random

- Rationale for selecting satellite groups:
N/A

- Post-exposure recovery period in satellite groups:
N/A

- Section schedule rationale (if not random):
Random

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes

- Chronological Sequence of Study:
Dose Groups 1 to 4
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) 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.
v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
ix) Blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

DETAILED CLINICAL OBSERVATIONS:

Clinical Observations:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Functional Observations:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

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

FOOD CONSUMPTION:
Yes.

- During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
Yes.
- Food efficiency:
Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during the pre-maturation phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation

WATER CONSUMPTION:
Yes.

- Time schedule for examinations:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION:
No
- Time schedule for examinations:
Not applicable

- Dose groups that were examined:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Yes.

- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and on Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

- Anaesthetic used for blood collection:
No

- Animals fasted:
No

- How many animals:
Five males and 5 females selected from each test and control group on Day 14.
Five males and 5 females selected from each surviving treatment group on Day 42
And surviving 500 mg/kg/day on Day 41

- Parameters checked in tables 18 -21 which are attached were examined.

URINALYSIS: No
- Time schedule for collection of urine:
Not applicable

- Metabolism cages used for collection of urine:
Not applicable

- Animals fasted:
Not applicable

- Parameters examined: Not applicable

NEUROBEHAVIOURAL EXAMINATION:
Yes.

(Functional Performance Tests)
Final week of treatment.

(Sensory Reactivity)
Final week of treatment.

- Dose groups that were examined:
(Behavioural assessment)
All animals

(Functional Performance Tests)
Five selected males and females per dose level

(Sensory Reactivity)
Five selected males and females per dose level

- Parameters examined:
(Behavioural assessment)
Detailed individual clinical observations were performed for each animal using a purpose-built arena.
The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation

(Functional Performance Tests)
Motor Activity:
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength:
An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

(Sensory Reactivity)
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach

OTHER:
MATING
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. 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 the oestrous cycle 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) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii)Date and time of observed start of parturition
iv)Date and time of observed completion of parturition


LITTER SIZE
On completion of parturition (Day 0 of 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:
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii)Sex of offspring on Day 1 and 4 post partum
iv)Clinical condition of offspring from birth to Day 5 post partum
v)Individual offspring and litter weights on Day 1 and 4 post partum


PHYSICAL DEVELOPMENT
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:testis During histopathology, the male epididymides were examined for spermatocoel granuloma formation.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring and litter weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS:
Dying offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals:
Adult surviving males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.

- Maternal animals:
Adult surviving females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY / ORGAN WEIGHTS

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).
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY

The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation. Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid and liver.

The following reproductive organs were weighed from all animals that were killed at the end of the study: ovaries, epididymides and testes.

Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin except where indicated.

Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi)(lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), mammarygland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), mid thoracic and lumbar, spleen, stomach, thyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), thymus, urinary bladder, uterus/cervix and vagina.

The following tissues were also removed from the remaining animals:coagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix.

All tissues were despatched to the Test Site Harlan Laboratories Ltd., Zelgliweg 1, CH-4452 Itingen, Switzerland for processing (Principal Investigator: K Weber). The tissues from five selected control and 200 mg/kg/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination.

The following tissues,Coagulation gland, Epididymides, Mammary glands, Ovarie, Pituitary, Prostate, Seminal vesicles, Testes, Uterus/Cervix and Vagina from the remaining control and 200 mg/kg/day were also processed. In addition, sections of testes and epididymides from all control and 200 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment-related changes in the stomach, examination was subsequently extended to include similarly prepared sections of the stomach from five animals per sex from the low and intermediate groups.
Microscopic examination was conducted by the Study Pathologist.

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone and all Necropsy findings recorded. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Haematology, blood chemistry, adult absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
The following statistical procedures were used:
For males and females prior to pairing and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The 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 showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
In addition, histopathological findings were analysed using the following methods:

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 paired ÷ Number of animals mated) x 100
Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) x 100
Gestation and Parturition Data
The following parameters were calculated for 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 pregnant females ÷ Number of females delivering live offspring) x 100

Offspring viability indices:

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 Corpora Lutea) ÷ Number of implantation sites] x 100
% post – implantation loss =[(Number of implantation sites - Number of implantation sites) ÷ Total number of offspring born] x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring born ÷Number of offspring alive on Day 1) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 1 ÷ Number of offspring alive on Day 4) x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 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:

effects observed, treatment-related

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:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
(Clinical signs)
Increased salivation was detected for animals of either sex from all treatment groups throughout the treatment period. Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test material formulation.
The female treated with 200 mg/kg/day that was found dead on Day 5 did not show any clinical signs of toxicity prior to death.
Remaining clinical observations were confined to staining around the snout or mouth in one male treated with 200 mg/kg/day on Day 19 and one male treated with 50 mg/kg/day on Day 41, an isolated incident of generalised scab formation in one male treated with 100 mg/kg/day between Days 19 and 21, hunched posture in one male treated with 50 mg/kg/day, generalised fur loss in one female treated with 200 mg/kg/day and noisy respiration in one female treated with 200 mg/kg/day on Day 28. In the absence of true dose related responses these incidences in isolation were not considered to be of toxicological significance.

(Mortality)
One female treated with 200 mg/kg/day was found dead on Day 5. Histopathological evaluation did not reveal the cause of this death.
There were no further unscheduled deaths during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
(Body weight)
Males treated with 200 mg/kg/day showed a statistically significant reduction in bodyweight gain (P<0.01), with the majority of males actually showing bodyweight losses during Week 1. Males from this treatment group continued to show a statistically significant reduction in bodyweight gain during Week 2. Females treated with 200 mg/kg/day showed actual bodyweight losses during the first week of treatment however statistical significance was not achieved.
No such effects were detected in animals of either sex treated with 100 or 50 mg/kg/day.

(Food consumption)
No adverse effect on food consumption was detected for males during the treatment period or for females during the pre-mating, gestation or lactation phases of the study. Food efficiency (the ratio of bodyweight gain to dietary intake) was however affected for males treated with 200 mg/kg/day during the first two weeks of treatment and for females from this treatment group during the first week of treatment.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No toxicologically significant effects in the reproductive parameters were observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment-related changes were reported.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
(Mating)
There were no treatment-related effects on mating or conception rates for treated animals.
One control and two females treated with 200 mg/kg/day failed to show any positive evidence of mating but subsequently gave birth to live young.
(Fertility)
No treatment-related effects were detected on fertility for treated animals when compared to controls.
All control, intermediate and high dose females were pregnant. One female treated with 50 mg/kg/day did not achieve pregnancy. In the absence of a true dose related response this intergroup difference was considered not to be of toxicological importance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant effects were detected in the organ weights measured.
Males treated with 200 mg/kg/day showed a statistically significant reduction in epididymides and thyroid weight both absolute and relative to terminal bodyweight, a statistically significant reduction in absolute liver weight and a statistically significant increase in relative liver weight. Males treated with 100 mg/kg/day also showed a statistically significant reduction in absolute and relative thyroid weight. In the absence of any associated histology correlates the intergroup difference was considered to be of no toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects were detected in treated terminal kill animals.
The female treated with 200 mg/kg/day that was found dead on Day 5 did not show any macroscopic abnormalities at necropsy. Histopathological evaluations did not reveal a cause of death for this animal.
One control female had white coloured fluid in the right kidney at necropsy. In the absence of treatment this was considered of no toxicological significance

HISTOPATHOLOGY (PARENTAL ANIMALS)
The following treatment-related effects were detected:
STOMACH: Hyperkeratosis of the forestomach was evident in animals of either sex treated with 200 and 100 mg/kg/day. Ulceration was also noted in one male and two females treated with 200 mg/kg/day. Further changes in the forestomach were identified as erosion in one female treated with 200 or 100 mg/kg/day, abscesses in the muscular layer in one male treated with 200 mg/kg/day and inflammation in the muscular layer in one male treated with 100 mg/kg/day and one female treated with 200 mg/kg/day.
The findings in the forestomach (hyperkeratosis, ulceration/erosion, inflammation/ abscesses) are considered to represent a local irritant effect of the test material.

SPLEEN: Not reported.

KIDNEY: One control female had white coloured fluid in the right kidney at necropsy. In the absence of treatment this was considered of no toxicological significance.

OTHER FINDINGS (PARENTAL ANIMALS)
WATER CONSUMPTION
No treatment-related intergroup differences in water intake were detected for treated animals when compared to controls.

HAEMATOLOGY
No toxicologically significant effects were detected in the haematological parameters examined.
Haematological examinations following termination of treatment revealed statistically significant reductions in mean cell haemoglobin concentration and clotting time and a statistically significant increase in haematocrit in males treated with 200 mg/kg/day. Females from all treatment groups showed a statistically significant reduction in mean cell haemoglobin concentration. All individual values were within the normal ranges for rats of the strain and age used and the intergroup differences were considered not to be of toxicological significance. Females treated with 200 and 100 mg/kg/day showed a statistically significant reduction in activated partial thromboplastin time. In the absence of a true dose-related response the intergroup differences were considered not to be of toxicological significance. Males treated with 200 mg/kg/day showed a statistically significant increase in monocytes and eosinophils. In the absence of any associated changes the intergroup differences were considered to be of no toxicological importance.

CLINICAL CHEMISTRY
No toxicologically significant effects were detected in the blood chemical parameters examined.
Animals of either sex treated with 200 mg/kg/day showed a statistically significant increase in albumin/globulin ratio. All individual values were within the normal range for rats of the strain and age used and the intergroup differences were considered not to be of toxicological significance. Males treated with 200 mg/kg/day showed a statistically significant reduction in alkaline phosphatase. The majority of individual values were within the normal range for rats of the strain and age used and a reduction in this enzyme is generally considered not to be of toxicological importance. Females treated with 100 mg/kg/day showed a statistically significant reduction in chloride concentration. In the absence of a dose related response the intergroup difference was considered to be of no toxicological importance.

NEUROBEHAVIOUR
(Behavioural assessment)
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
(Functional Performance Tests)
There were no toxicologically significant changes in functional performance.
Females treated with 50 mg/kg/day showed a statistically significant reduction in mean hind limb grip strength. This intergroup difference was confined to one out of the three tests and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.

(Sensory Reactivity)
There were no treatment-related changes in sensory reactivity.

Uterine Examination.
No treatment related effects reported.

Gestation Length
No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22 to 23½ days.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Reproductive Toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The oral administration of Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane to rats by gavage, at dose levels of 200, 100 and 50 mg/kg/day, resulted in treatment-related effects at 200 and 100 mg/kg/day

Remarks on result:

other: Generation: Parents and offsprings (migrated information)

2

Dose descriptor:

NOEL

Remarks:

Systemic Toxicity

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)

Offspring Litter Size and Viability. There was no adverse effect on litter size for treated animals when compared to controls and there was no obvious difference in sex ratio for litters from treated females in comparison to controls.

CLINICAL SIGNS (OFFSPRING)
Of the four litters produced at 500 mg/kg/day, one litter had one dead offspring. No abnormalities were detected in the remaining animals. Another litter had one dead offspring and four of the remaining offspring were cold and weak. All the animals in a further litter were cold. The whole of the remaining litter were cold with no milk in the stomach, they were also weak and two pups were found dead and five were missing.
Of the nine pregnancies at 150 mg/kg/day one litter had a number of dead offspring and the remaining offspring and dam were of poor physical health resulting in their termination.
The incidental findings detected in the control, 50 and remaining 150 mg/kg/day dose group were considered to be incidental.


BODY WEIGHT (OFFSPRING)
There was no significant intergroup difference in mean litter weights or offspring bodyweights for treated animals in comparison to controls.

SEXUAL MATURATION (OFFSPRING)
Not applicable

ORGAN WEIGHTS (OFFSPRING)
Not applicable

GROSS PATHOLOGY (OFFSPRING)
Offspring: Two females treated with 500 mg/kg/day produced a litter with one death at birth. One pup displayed autolytic changes and one pup had no milk in the stomach. On Day 2 post partum three pups in one 150 mg/kg/day and two pups in one 50 mg/kg/day litter were found dead. These pups displayed autolytic changes.
The incidental signs detected in litters from the remaining dose groups were of no toxicological importance.


HISTOPATHOLOGY (OFFSPRING)
As the 500 mg/kg/day treatment group was terminated early due to excessive toxicity no definitive reproductive effect could be established. However there was no effect on pre-natal development in comparison to controls.
No effect of treatment was detected on reproduction or offspring development, at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for reproductive and development toxicity was considered to be 150 mg/kg/day.


OTHER FINDINGS (OFFSPRING)
Offspring Growth and Development:
There was no significant intergroup difference in mean litter weights and offspring bodyweights for treated animals in comparison to controls.
A reduction in surface righting was evident in the 500 mg/kg/day offspring in comparison to controls. This was considered to be attributed to maternal nurturing.
No such effect was detected at 150 and 50 mg/kg/day.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Histopathology

Incidence and mean severity of main findings in the forestomach.

Finding / Groups Total examined	1 (5) M        (5) F		2  (5) M       (5) F		3  (5) M       (5) F		4 (10) M        (9) F
Affected / Mean Severity
Ulceration	0	0	0	0	0	0	1/3.0	2/2.0
Erosion	0	0	0	0	0	1/1.0	0	1/2.0
Abscess	0	0	0	0	0	0	1/3.0	0
Hyperkeratosis	0	0	0	0	1/2.0	8/1.5	1/1.0	8/2.0
Inflammation	0	0	0	0	1/2.0	0	0	1/2.0

All other findings noted were considered to be incidental findings commonly noted in animals of this strain and age.

Applicant's summary and conclusion

Conclusions:

The oral administration of Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane to rats by gavage, at dose levels of 200, 100 and 50 mg/kg/day, resulted in treatment-related effects at 200 and 100 mg/kg/day. No such effects were detected at 50 mg/kg/day therefore the 'No Observed Effect Level' (NOEL) for systemic toxicity was considered to be 50 mg/kg/day.
No adverse treatment-related effects were observed for reproduction, therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 200 mg/kg/day.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (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 material was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 50, 100 and 200 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated at termination on five selected males and females from each dose group. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Mortality.One female treated with 200 mg/kg/day was found dead on Day 5. There were no further unscheduled deaths during the study.

Clinical Observations.Animals of either sex from all treatment groups showed episodes of increased salivation immediately post or one hour post dosing throughout the treatment period.

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

Bodyweight.Animals of either sex treated with 200 mg/kg/day showed actual bodyweight losses during Week 1 of treatment. Males from this treatment group continued to show a reduction in bodyweight gain during Week 2. Females treated with 200 mg/kg/day showed actual bodyweight losses during the first week of treatment.

No such effects were detected in animals of either sex treated with 100 or 50 mg/kg/day.

Food Consumption.No adverse effect on food consumption was detected. Food efficiency was however reduced for males treated with 200 mg/kg/day during the first two weeks of treatment and for females from this treatment group during the first week of treatment. 

No such effects in food efficiency were detected in animals of either sex treated with 100 or 50 mg/kg/day.

Water Consumption.No intergroup differences were detected.

Haematology.No toxicologically significant effects were detected in the haematological parameters measured.

Blood Chemistry.No toxicologically significant effects were detected in the blood chemical parameters measured.

Reproductive Performance:

Mating and Fertility.There were no treatment-related effects on mating or conception rates for treated animals.

One female treated with 50 mg/kg/day was found to be non-pregnant. One control and two females treated with 200 mg/kg/day failed to show any positive evidence of mating but subsequently gave birth to live young.

Gestation Length.There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size and Viability.Of the litters born, litter size at birth and subsequently on Days 1 and 4post partumwere comparable to controls.

Offspring Growth and Development.Litter weights at Day 4post partumwere reduced in females treated with 200 mg/kg/day.

No such effects were detected in litters from females treated with 100 or 50 mg/kg/day.

Litter observations.No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Necropsy.No toxicologically significant effects were detected.

Histopathology.The following treatment-related effects were detected:

STOMACH:Hyperkeratosis of the forestomach was evident in animals of either sex treated with 200 and 100 mg/kg/day. Ulceration was also noted in one male and two females treated with 200 mg/kg/day. Further changes in the forestomach were identified as erosion in one female treated with 200 or 100 mg/kg/day, abscesses in the muscular layer in one male treated with 200 mg/kg/day and inflammation in the muscular layer in one male treated with 100 mg/kg/day and one female treated with 200 mg/kg/day.

Conclusion.The oral administration of Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane to rats by gavage, at dose levels of 200, 100 and 50 mg/kg/day, resulted in treatment-related effects at 200 and 100 mg/kg/day. No such effects were detected at 50 mg/kg/day therefore the 'No Observed Effect Level' (NOEL) for systemic toxicity was considered to be 50 mg/kg/day.

No adverse treatment-related effects were observed for reproduction, therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 200 mg/kg/day.