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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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:

other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/09/2009 Date of signature: 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

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

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

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)

- 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

Duration of treatment / exposure:

Up to 54 consecutive days.

Frequency of treatment:

Daily

Duration of test:

Up to 54 consecutive days.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day (0 mg/l)
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:
The dose levels were chosen based on the results of a Seven Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Harlan Laboratories Ltd Study Report No. 0044-1419)

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

- Other:
Not applicable

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule:
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).

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule:
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).

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 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. Weekly food consumptions were performed weekly for each cage of adults throughout the study period.

FOOD EFFICIENCY:
Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males and females throughout the study period.

WATER CONSUMPTION: Yes
- Time schedule for examinations:Water intake was observed daily by visual inspection of water bottles for any overt
changes.
A possible treatment-related effect was detected on the range finder animals, therefore gravimetric measurements were initiated from Day 1 throughout to study termination.

POST-MORTEM EXAMINATIONS: Yes
- 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.
The 500 mg/kg/day treatment group was considered to be excessive and terminated on Day 41.

- Organs examined:
Gross pathology: For all females the uterus was examined for signs of implantation and the number of uterine implantations in each born was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. 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/organ weights:
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), mammary gland, 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 Harlan Laboratories Ltd, Switzerland (Principal Investigator: K Weber). The tissues from five selected control, 150 and 500 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control, 150 and 500 mg/kg/day were also processed. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidney and spleen from five animals per sex from the low dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

OTHER:
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 on Day 14 (day prior to pairing) and five males and five females selected from each surviving treatment group on Day 42. Blood samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at Days 41 and 42.

- Anaesthetic used for blood collection:
Not stated in the report

- 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

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


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:

(Behavioural assessment)
Prior to the start of treatment at weekly intervals thereafter

(Functional Performance Tests)
Prior to termination

(Sensory Reactivity)
Prior to termination

- 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).

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.

(Sensory Reactivity)
Animals were assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach


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 mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Estrous cyclicity (Parental)
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:During histopathology, the male epididymides were examined for spermatocoel granuloma formation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
Yes
Examinations included:
- Gravid uterus weight:
No

- Number of corpora lutea:
Yes

- Number of implantations:
Yes

- Number of early resorptions:
No

- Number of late resorptions:
No

- Other:

Fetal examinations:

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 and dead offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

POST-MORTEM EXAMINATION
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 28 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities wererecorded.

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, absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights
The following statistical procedures were used:
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.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and
landmark developmental markers.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Historical control data:

Not applicable.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
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

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

2

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The following assessment of litter response is generally based on those litters reared to termination on Day 5 post partum, although data available for females showing total litter loss has also been taken into consideration, where considered appropriate.

VIABILITY (OFFSPRING)
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.

CLINICAL SIGNS (OFFSPRING)
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.


BODY WEIGHT (OFFSPRING)
Not applicable

ORGAN WEIGHTS (OFFSPRING)
Not applicable.

GROSS PATHOLOGY (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
HISTOPATHOLOGY (OFFSPRING)
Not examined.

OTHER FINDINGS (OFFSPRING)
Not applicable.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Histopathology

The findings in the forestomach (hyperkeratosis, ulceration/erosion, inflammation/ abscesses) are considered to represent a local irritant effect of the test material.

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:

CONCLUSION
The oral administration of Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane to rats for a period of forty-two days for males and up to fifty-four days for females (including two weeks pre-mating, gestation and early lactation period) at dose levels of up to 200 mg/kg/day resulted in no toxicologically significant effects in the reproductive and developmental parameters which were measured during the course of the study. , As no adverse treatment-related effects were observed for reproduction/development , a ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive/developmental 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 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 4 post partum were 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 for a period of forty-two days for males and up to fifty-four days for females (including two weeks pre-mating, gestation and early lactation period) at dose levels of up to 200 mg/kg/day resulted in no toxicologically significant effects in the reproductive and developmental parameters which were measured during the course of the study. , As no adverse treatment-related effects were observed for reproduction/development , a ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive/developmental toxicity was considered to be 200 mg/kg/day.