1,2-Cyclohexanedicarboxylic acid, 1-[2-[(1-oxo-2-propen-1-yl)oxy]ethyl] ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from similar mixture/product

Adequacy of study:

key study

Study period:

13 September 2011 and 16 March 2011

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 relevant results.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
(See the attached report in section 13.2)
In line with the read across justification for the repeated dose toxicity test, the reproduction-developmental toxicity test with MAES can be used to assess the possible reproductive toxicity of MAHP. Also complying with the animal control act, the use of this study is justified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 20/07/2010 Date of signature: 29/10/2010

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

MAES

IUPAC Name:

MAES

Constituent 2

Reference substance name:

-

EC Number:

478-130-6

EC Name:

-

Cas Number:

50940-49-3

Molecular formula:

C9H12O6

IUPAC Name:

2-(acryloyloxy)ethyl hydrogen succinate

Details on test material:

- Name of test material (as cited in study report): MAES
- Substance type: Organic
- Physical state: Extremely pale yellow liquid
- Analytical purity: 95.50 %
- Lot/batch No.: Not advised
- Label : MAES Monomer
- Expiration date of the lot/batch: Not advised
- Date received : 05 August 2010
- Storage conditions : room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST strain rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) ~12 wks
- Weight at study initiation: (P) Males: 327-393 g; Females: 188-231 g
- Housing:
Pre-mating: groups of 5 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
Mating: One male, One female in polypropylene grid cages suspended over trays lined with absorbent paper.
Post-mating: Males returned to original cages, females in individual solid floor polypropylene cages with stainless steel mesh lids and softwo od flake bedding
- Diet: ad libitum access to A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used
- Water: ad libitum access to mains drinking water in polycarbonate bottles. The drinking water was not considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 September 2010 (first day of treatment) - 07 November 2010 (final necropsy day)

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

other: polyethylene glycol (400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 2251-0026). Results from the previous study showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark. Samples of each test item formulation were taken and analysed for concentration of MAES at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix (chemistry report needs to be attached as an appendix). The results indicate that the prepared formulations were within 5% of the nominal concentration and considered acceptable for the purpose of this study.

DIET PREPARATION
Not applicable

- Rate of preparation of diet (frequency):
Not applicable

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

- Storage temperature of food:
No data

VEHICLE
Polyethylene glycol 400.

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

- Concentration in vehicle:
0, 3.75, 12.5 and 37.5 mg/ml

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

- Lot/batch no. (if required):
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: Until mating, or up to 14 days maximum
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for t e 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 sp rm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).

- After successful mating each pregnant female was caged individually during the period of gestation and lactation.

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
Not applicable

- Further matings after two unsuccessful attempts:
Not applicable

- Any other deviations from standard study plan:
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity, stability and linearity determinations were performed under Harlan Laboratories Ltd project number 2251-0026.
The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
The concentration of the test material in the formulations was determined using high performance liquid chromatography (HPLC). The test item formulations were diluted with acetonitrile to give a final, theoretical test item concentration of approximately 0.1 mg/ml. Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 0.1mg/ml.

The test item formulations were sampled and analysed within three days of preparation.
The results indicate that the prepared formulations were acceptable for the purpose of this study.

Duration of treatment / exposure:

All males from all treatment groups were terminated on Day 43, followed by the termination of all 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.

Frequency of treatment:

Daily

Details on study schedule:

Ten male and ten female animals were treated daily at the appropriate dose level throughout the study. The first day of dosing was designated as Day 1 of the study.
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
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.

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.

Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested
3.75 mg/ml

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150mg/kg/day
Basis:
actual ingested
37.5mg/ml

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of previous toxicity testing of this chemical.
- Rationale for animal assignment (if not random): Randomized based on stratified body weights to ensure similarities of all dose groups.
- Other: All animals uniquely identified by ear punch.

Positive control:

Not applicable

Examinations

Parental animals: Observations and 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, thirty minutes after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes (see above).
- Yes

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

FOOD CONSUMPTION:
- 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.

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

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes. Intergroup differences did not indicate any need for more formal gravimetric measurements.

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:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this
data)

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

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: testes and epididymides during histopathology.

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: Males were teminated following successful second mating with untreated females.

- Maternal animals: All treated females and offspring were killed on day 5 post partum and untreated females were killed on Day 15 post coitum.

GROSS NECROPSY

- Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination after the successful re-mating. Adult treated females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of odium pentobarbitone on Day 5 post partum. Untreated females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 15 post coitum.

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.

In addition, the corpora lutea of all ovaries and uterine implantation sites fromfrom pregnant females were counted at necropsy. The procedure for counting implantation sites was enhanced (where necesary) by staining the uteri with a 1% ammonium polysulphide solution.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [23 for males and 24 for females] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY
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.

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

Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Coagulating gland
Prostate
Epididymides*
Seminal vesicles
Ovaries
Testes*
Mammary gland (females only)
Uterus/Cervix
Pituitary
Vagina

All tissues were dispatched to Harlan Laboratories Ltd, Switzerland

The tissues from control and 150 mg/kg/day dose group animals, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes and epididymides from all control and 150 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Statistics:

The following parameters were subjected to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight relative organ weights (Males)

The following statistical procedures were used:
For males and females during the pre-mating phase, 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 Bartletts’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 nonhomogeneity 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 tes s 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. Non-parametric methods were also used to analyse implantation loss, offspring sex ratio.

Reproductive indices:

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 (%) = x 100

Pregnancy Index (%) = 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 (%) = 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:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No findings in reproductive organs

Histopathological findings: neoplastic:

not examined

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)

No clinical signs of systemic toxicity or indications of treatment-related behavioural
change were observed.
Animals of either sex treated with 150 mg/kg/day showed episodes of increased salivation around the time of dosing from Day 2 through to Day 42. A similar finding was evident intermittently in males treated with 50 mg/kg/day between Day 14 and Day 40.
Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test item formulation and in the absence of supporting evidence of toxicity is considered not to be indicative of systemic toxicity. One instance of transient increased salivation was observed in one control male.
Additional findings identified (also associated with vehicle/test formulation palatability/irritancy) included sporadic instances of staining around the mouth, noisy respiration and sneezing (latter at 50 mg/kg/day in one male only) were evident on occasion in both test and controls group animals during the study.
One instance of generalised fur loss was noted in one 15 mg/kg/day female between Day 41 and 45. Such changes are occasionally seen in pregnant female rats and are considered not to be related to test item toxicity.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean litter weights and body weight gains were considered to have been unaffected by maternal exposure.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The percentage of offspring who successfully showed surface righting reflex on Day 1 and the type, incidence and distribution of clinical signs in the offspring to termination on Day 5 of age were unaffected by maternal exposure.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

other: righting reflex

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of MAES to rats by gavage at dose levels of 15, 50 and 150 mg/kg/day resulted in treatment-related effects associated with the locally irritant properties of the test item and on this basis a “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity, was not established in this study.
The above findings under the conditions of this study were shown to have no adverse impact on reproductive performance and for this reason the No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 150 mg/kg/day.

Executive summary:

Introduction.The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study is compatible with the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible 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 item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 15, 50 and 150 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 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. Adult males were terminated on Day 43, and all females with offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results.

Adult Responses:

Mortality.There were no unscheduled deaths.

Clinical Observations.No clinical signs of toxicity or indications of treatment-related behavioural change were observed.

Animals of either sex treated with 150 mg/kg/day showed transient episodes of increased salivation around the time of dosing from Day 2 onwards. A similar finding was evident intermittently in males treated with 50 mg/kg/day from Week 2 onwards. Sporadic instances of staining around the mouth, noisy respiration and sneezing were also observed in test and control group animals during the study. Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test item formulation and in the absence of supporting evidence of toxicity are considered not to be indicative of systemic toxicity.

Body Weight.Males treated with 150 mg/kg/day showed a statistically significant decrease in body weight gain at Week 2 only in comparison with the concurrent controls. No such effects were detected in females treated with 150 mg/kg/day or in animals of

either sex treated with 15 and 50 mg/kg/day.

Food Consumption and Food Efficiency.No adverse effects on food consumption or food efficiency were detected for treated animals when compared with controls.

Water Consumption.No intergroup differences were detected.

Reproductive Screening:

Mating.There were no treatment-related effects on mating in males or females treated with 15, 50 or 150 mg/kg/day.

Fertility.There were no treatment-related effects detected in conception rates. No treatment-related effects on fertility were detected between treated animals when compared to controls.

Gestation Lengths.No treatment-related effects were detected for the length of gestation between control and treated groups. The length of gestation ranged between 22 to 24 days for control and test animals. Statistical analysis of the data did not reveal any significant intergroup differences.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. No significant differences in litter size, viability or sex ratio were evident for offspring from treated litters when compared to those from the controls.

Offspring Growth and Development.Offspring body weight gain and litter weights at birth and subsequently on Day 1 and Day 4 post partum were comparable to controls. No adverse effects were detected in surface righting reflex on Day 1.

Offspring Observations.No clinical signs of toxicity or indications of treatment-related behavioural change were observed for offspring from all treatment groups.

Pathology:

Necropsy.No treatment-related macroscopic findings were observed in the reproductive organs.Treatment-related gastric inflammation identified as a raised limiting ridge and thickening of the glandular gastric epithelium were identified in three males and one female at 15 mg/kg/day, five males and one female at 50 mg/kg/day and in nine males and two females at 150 mg/kg/day. In addition the glandular region of the stomach of two150 mg/kg/day males showed reddened patches.

Organ Weights.No treatment-related effects were detected in the organ weights measured.

Histopathology.There were no treatment related histopathological changes detected in the reproductive organs. However, treatment-related findings characterized by minimal to slight gastric irritation were detected in the stomach of three males and one female at 15 mg/kg/day, five males and one female at 50 mg/kg/day and nine males and two females at 150 mg/kg/day.

Conclusion.The oral administration of MAES to rats by gavage at dose levels of 15, 50 and 150 mg/kg/day resulted in treatment-related effects associated with the locally irritant properties of the test item and on this basis a “No Observed Adverse Effect Level”

(NOAEL) for systemic toxicity, was not established in this study. The above findings under the conditions of this study were shown to have no adverse impact on reproductive performance and for this reason the No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 150 mg/kg/day.