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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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:

2010

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

Vehicle:

arachis oil

Details on oral 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.

VEHICLE
Arachis oil

- Justification for use and choice of vehicle (if other than water):
See analytical results

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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.

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:

Fifty-four consecutive days

Frequency of treatment:

Daily

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 (50 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)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

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

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

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

URINALYSIS: No

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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. 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 26 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).
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 following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Prostate Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix).


HISTOPATHOLOGY: Yes.
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin except where stated:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate Brain (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulation gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skin (hind limb), Eyes, Spinal cord (cervical, mid-thoracic and Gross lesions lumbar), Heart, Spleen, Ileum, Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes, Lungs (with bronchi) , Thymus, Lymph nodes (cervical and mesenteric)Urinary bladder, Mammary gland , Uterus/Cervix, Muscle (skeletal), Vagina.

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. 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.

Other examinations:

None.

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. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

non-adverse

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

ADULT RESPONSES

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 WEIGHT GAIN
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 AND FOOD EFFICIENCY
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.

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.


ORGAN WEIGHTS
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 19 (statistically significant differences are indicated). Individual data are given in Appendix 19 and Appendix 20.
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
Adults: One 500 mg/kg/day male showed small seminal vesicles, whilst another, showed a mass attached to the mandibular lymph nodes. In addition three females from this treatment group had dark contents of the gastrointestinal tract, two of these also showed a distended stomach. A dark content of the stomach was seen in two of the females with enlarged adrenals also noted in one of these. One female showed a dark mass on the left horn of the uterus.
A yellow fluid filled mass on the right epididymis was evident in one 50 mg/kg/day male. Two females treated with 50 mg/kg/day showed epithelial sloughing of the glandular region of the stomach, whilst two additional females showed epithelial sloughing of both regions of the stomach. In addition, reddened lungs were seen in one 50 mg/kg/day female.
Epithelial sloughing of the glandular region of the stomach was seen in two control females one of which also showed both ovaries encased in a clear fluid filled sac. One additional control female displayed dark viscous fluid filled uterus.
One interim death male treated with 500 mg/kg/day showed a distended stomach with autolysis of the gastrointestinal tract and small seminal vesicles. One male showed distended stomach with fluid filled dark viscous contents and a small caecum. In addition, one interim death female treated with 500 mg/kg/day was found partially cannibalised, with enlarged adrenals and an enlarged stomach, with dark contents. A distended stomach, with autolysis of the gastrointestinal tract was seen in another interim death female. Dark coloured contents of the stomach and enlarged adrenals were evident in the remaining interim death females treated with 500 mg/kg/day. One also showed dark contents of the gastrointestinal tract.
One interim death female treated with 150 mg/kg/day showed dark coloured contents of the stomach with the remaining gastrointestinal tract empty.

Offspring
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.

Adults
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: NON-NEOPLASTIC
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.

URINALYSIS
Not examined

OTHER FINDINGS
Reproductive Performance:

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.

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.

Litter response
In total, ten females from the control and 100 mg/kg/day dose groups and nine females from the 50 and 200 mg/kg/day dose group gave birth to a live litter and successfully reared young to Day 5 of age.
One non-pregnant female was observed in the 50 mg/kg/day dose group. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size and Viability
No toxicologically significant effects were detected.
No significant differences were detected for corpora lutea and implantation counts for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
An increase in pre implantation losses was evident at 200 mg/kg/day together with a reduction in litter size at birth, Day 1 and Day 4. Statistical significance was not achieved in either parameter. The intergroup differences were considered to be an inconclusive effect that was due to three females each showing an isolated individual effect that was outside of the normal group distribution and as such was considered not to represent a true indication of test material toxicity.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Offspring Growth and Development
No toxicologically significant effects were detected.
A statistically significant reduction in litter weight was evident at 200 mg/kg/day. This was considered to be a consequence of the inconclusive isolated effects seen in three females in this treatment group and in the absence of an effect on mean offspring bodyweights the intergroup difference was considered not to be a true indication of test material toxicity.
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, cold, weak, no milk in stomach, scattered and pale, were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test material toxicity.
No treatment-related effects were detected for surface righting reflex for offspring from treated animals when compared to offspring from control females. Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

Reproductive Toxicity

Effect level:

200 other: mg/kg/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

2

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

50 other: mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Histopathology

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 characterized by hyperkeratosis, ulceration/erosion and inflammation/abscesses of the forestomach resulting from local irritation at the site of contact during gavage dosing . No such effects were detected at 50 mg/kg/day. Other than local irritation in the stomach there was no evidence of toxicity at any treatment level. Based on the data the 'No Observed Effect Level' (NOEL) for local (site of contact) irritation is 50 mg/kg/day and in the absence of any other toxicological effects the the 'No Observed Effect Level' (NOEL) for systemic 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 4post partum. Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post 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 characterized by hyperkeratosis, ulceration/erosion and inflammation/abscesses of the forestomach resulting from local irritation at the site of contact during gavage dosing . No such effects were detected at 50 mg/kg/day. Other than local irritation in the stomach there was no evidence of toxicity at any treatment level. Based on the data the 'No Observed Effect Level' (NOEL) for local (site of contact) irritation is 50 mg/kg/day and in the absence of any other toxicological effects the the 'No Observed Effect Level' (NOEL) for systemic toxicity was considered to be 200 mg/kg/day.