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

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

other: review available data

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

The evaluation of the toxicokinetic behaviour of mono-(2-acryloxyethyl)hexahydrophthalate (MAHP) is based on toxicity studies performed with MAHP (acute oral) or with the analogue monoacryloyloxyethyl Succinate (MAES). The evaluation is put in a separate report included in the dossier (section 13.2). This robust summary contains parts of this evaluation related to the ADME endpoints.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

exposure-related information

Reference

The acute oral toxicity of the test material in rat was tested at 300 and 2000 mg/kg bw. The highest dose group was tested twice in three female rats. Since no mortality or sub-lethal effects were observed, the acute oral median lethal dose (LD50) was estimated to be greater than 2500 mg/kg bw.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 500 mg/kg bw

Quality of whole database:

Study according to OECD guideline under GLP: LD0 ≥ 2000 mg/kg bw.

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Acute Oral Toxicity: The study was performed to assess the acute oral toxicity of the test material following a single oral administration in the Sprague-Dawley CD strain rat. The method was designed to meet the requirements of the following:

- OECD Guidelines for the Testing of Chemicals No. 423 "Acute Oral Toxicity - Acute Toxic Class Method" (adopted 17 December 2001).

- Method B1 tris Acute Toxicity (Oral) of Commission Directive 2004/73/EC

The test material was administered orally as a solution in arachis oil BP for the 300 mg/kg dose level and orally undiluted for the 2000 mg/kg dose level. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy.

There were no deaths.There were no signs of systemic toxicity. No abnormalities were noted at necropsy.

The acute oral median lethal dose (LD50) of the test material in the female Sprague-Dawley CD strain rat was estimated to be greater than 2500 mg/kg bodyweight (Globally Harmonised Classification System - Unclassified).

Since no mortality occurred exposing two groups of three rats to 2000 mg/kg bw the substance is expected to be classified as acute oral cat. 5 or unclassified.

Cross-reference 2

Reason / purpose for cross-reference:

exposure-related information

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from similar mixture/product

Adequacy of study:

key study

Study period:

11 April 2006 to 21 August 2006.

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.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
(See the attached report in section 13.2)
Based on the fact that both MAHP and MAES result in similar metabolites in rat after oral uptake, it is justified to use the repeated dose toxicity test in rats with MAES for determining the DNEL for MAHP. Additional justification lies in the fact that using the MAES study for MAHP significantly saves test animals. Since the molecular mass and overall structure of the two molecules are comparable, no correction is made for molecular mass difference (1.25 for MAHP versus MAES) to calculate the DNEL value.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Commission Directive 96/54/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 30 August 2005 Date of Signature: 21 November 2005)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, UK.
- Age at study initiation: 5 to 6 weeks old.
- Weight at study initiation: 211 - 238 g (male) and 143 - 180 g (female).
- Fasting period before study: Not reported.
- Housing: The animals were housed in groups of 5 by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet: Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK. Ad libitum.
- Water: Mains drinking water ad libitum.
- Acclimation period: 9 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): ≥ 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

IN-LIFE DATES: Day 0 (the day of dosing) up to Day 28.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at 3.75, 37.5 and 75 mg/ml, as a solution in polyethylene glycol 400. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations to be stable for at least 14 days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

DIET PREPARATION
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The stability and homogeneity of the test material formulations were determined at the test laboratory. The samples used were test material extracted with acetonitrile with final theoretical concentration of 0.1 mg/ml (see 'Details on analytical verification of doses or concentrations'). The formulations were found to be stable for at least 14 days.
- Amount of vehicle (if gavage): 4 ml/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary; The concentration of MAES in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Samples; The test material formulations were diluted with acetonitrile to give a final theoretical test material concentration of 0.1 mg/ml.

Standards; Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.

Procedure; The sample and standard solutions were analysed by HPLC.

Homogeneity determinations; The test material formulations were mixed throughly and samples were taken from the top, middle and bottom of the container, shaking between sampling in triplicate.

Stability determinations; The test material formulations were sampled and analysed initially and then after storage at 4ºC in the dark for 14 days.

Verification of test material formulation concentrations; The test material formulations were sampled and analysed within 3 days of preparation.

Results; Mean concentration found was in the range of 93 – 101% of nominal concentrations over the dosing period. The results showed the formulation to be stable for at least 14 days.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily.

Remarks:

Doses / Concentrations:
15 mg/kg/day
Basis:
other: nominal in vehicle

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
other: nominal in vehicle

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

5 animals per sex per dose and the control group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of the range-finding study performed. See the summary of the range-finding study in 'any other information on materials and methods incl. tables'.

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, immediately post dosing and 1 and 5 hours after dosing during the working week. Animals were observed immediately before dosing, and 1 hour after dosing at weekends and public holidays.
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and weekly intervals thereafter. Bodyweights were also recorded prior to terminal kill.

FOOD CONSUMPTION: Yes
Weekly food consumption was recorded for each cage group.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight gain / food consumption) was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily for each cage group by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on Day 28. Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No.
- How many animals: All surviving animals.
- Parameters checked in Tables 10 and 11.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Prior to the start of treatment and on Days 3, 10, 17 and 25, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Dose groups that were examined:
All animals.

- 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 of one sex were tested at each occasion and were randomly allocated to the activity monitors. The evaluation period was one hour for each animal. The time (seconds) each animal was active and mobile was recorded for the overall one hour 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: Each animal was individually 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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All suriviving animals were killed by intravenous overdose of sodium pentobarbitone, followed by exsanguination. All animals 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, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys and thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from all animals and preserved in buffered 10% formalin.
Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, 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), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

All tissues were despatched to PPropath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK. The tissues (except aorta, bone & bone marrow, eyes, muscle, oesophagus, pancreas, pituitary, salivary glands and skin) from all control and 300 mg/kg/day group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg/day dose group animals.

Since there were indications of treatment-related changes in the bone marrow, stomach and duodenum, examination was subsequently extended to include sections of these tissues from all animals in the remaining 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 examinations:

No.

Statistics:

All data was summarised in tabular form. 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 or ANCOVA and Bartlett'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).

Probability values (p) were calculated as follows:
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality: one female but this was not accompanied with any adverse clinical signs or macroscopic indication. Clinical signs: Considered to be related to the irritancy of the test substance administered.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality: one female but this was not accompanied with any adverse clinical signs or macroscopic indication. Clinical signs: Considered to be related to the irritancy of the test substance administered.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

non statistically significant changes.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

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:

MORTALITY
One female treated with 300 mg/kg/day was found dead on Day 4. There were no other unscheduled deaths.


CLINICAL SIGNS
See Table 1 for the results, as attached.
Animals of either sex treated with 300 mg/kg/day showed increased salivation around the time of dosing from Day 3 (males) and 7 (females) onwards. An isolated incident of noisy respiration was evident in one male and one female treated with 300 mg/kg/day. Red/brown staining around the mouth/snout was also evident in two animals treated with 300 mg/kg/day. One male treated with 150 mg/kg/day showed generalised fur loss and scab formation from Day 18 onwards. Animals of either sex treated with 150 and 15 mg/kg/day showed incidents of increased salivation around the time of dosing during the final two weeks of treatment. Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test material and, in isolation, are considered not to be indicative of systemic toxicity. The animal that died during the
study showed no adverse clinical signs prior to death.


BODY WEIGHT AND WEIGHT GAIN
See tables 6 and 7 for the results, as attached..
Animals of either sex treated with 300 mg/kg/day showed a reduction in bodyweight gain during Week 4 only; statistical significance however was not achieved. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.


FOOD CONSUMPTION AND FOOD EFFICIENCY
See tables 8 and 9 for the results, as attached..
Animals of either sex treated with 300 mg/kg/day showed a reduction in food efficiency during Week 4.
No such effects were detected in food efficiency for animals of either sex treated with 150 or 15 mg/kg/day or in food consumption for animals of either sex treated with 300, 150 or 15 g/kg/day.


WATER CONSUMPTION
Daily visual inspection of water bottles revealed no intergroup differences.


HAEMATOLOGY
See Table 10 for the results, as attached..
Animals of either sex treated with 300 mg/kg/day showed slight reductions in haemoglobin, haematocrit and erythrocyte count (statistical significance was however not always achieved).
Associated changes for males also included a statistically significant reduction in mean cell haemoglobin concentration.
No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.
Females from all treatment groups showed a statistically significant reduction in eosinophil count. In the absence of a dose related response these intergroup differences were considered not to be toxicologically significant.


CLINICAL CHEMISTRY
See Table 11 for the results, as attached..
There were no toxicologically significant changes in the blood chemical parameters measured.
Males treated with 300 and 150 mg/kg/day showed a statistically significant increase in plasma urea. All individual values were within the normal ranges for rats of the strain and age used, and in the absence of any histopathological evidence to suggest renal dysfunction, the increase was considered to be of no toxicological importance. Females from all treatment groups showed a statistically significant reduction in alanine aminotransferase. Reductions in plasma levels of this enzyme are unlikely to be of any toxicological significance, and with the majority of individual values within the normal range for rats of the strain and age used, the intergroup differences were considered to be of no toxicological importance.


NEUROBEHAVIOUR
See tables 2 - 5 for the results, as attached..
Behavioural Assessments
There were no treatment-related changes in the behavioural parameters measured.
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests
There were no treatment-related changes in the functional performance parameters measured.
Statistical analysis revealed no significant intergroup differences.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and was of no toxicological importance.


ORGAN WEIGHTS
See Table 12 for the results, as attached..
There were no treatment related changes in the organ weights measured.
Statistical analysis revealed no significant intergroup differences.


GROSS PATHOLOGY
See Table 13 for the results, as attached..
The female treated with 300 mg/kg/day that was found dead on Day 4 showed a dark liver and a raised limiting ridge in the stomach together with a pale and sloughing glandular gastric epithelium.
One male and four females treated with 300 mg/kg/day together with two males and one female treated with 150 mg/kg/day showed a raised limiting ridge in the stomach.
No such effects were detected in animals of either sex treated with 15 mg/kg/day.

One male treated with 300 mg/kg/day incurred a damaged eye during removal at necropsy. This was a physical injury occurring after termination and was considered unrelated to treatment. One female treated with 15 mg/kg/day showed red lungs at necropsy. In the absence of any histopathology correlates this intergroup difference was considered of no toxicological significance.


HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment-related microscopic changes were detected: See Table 14 for the results, as attached..
BONE MARROW: A lower incidence of higher grades of adipose infiltration of the bone
marrow, indicative of marrow hyperplasia, was observed in relation to treatment for females
treated with 300 mg/kg/day. This was a marginal effect that was dependent upon three animals at the high dose level having lower grades of severity. There was no convincing effect at any other dose level, or among males at any dose level.

STOMACH: Gastric changes characterised by acanthosis/hyperkeratosis of the forestomach and limiting ridge, mucosal hypertrophy, and mucosal basophilia were seen among animals of either sex treated with 300 mg/kg/day or at 150 mg/kg/day, but not at 15 mg/kg/day.

DUODENUM: Mucosal hypertrophy was seen in relation to treatment for males only treated with 300 mg/kg/day, 150 mg/kg/day, and at 15 mg/kg/day with an apparent dose response in terms of severity grades. However there were no associated degenerative changes. Females were not similarly affected.

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The oral administration of MAES to rats for a period of up to twenty-eight consecutive days at dose levels of 15, 150, and 300 mg/kg/day resulted in treatment-related effects in animals of either sex treated with 300 and 150 mg/kg/day and in males treated with 15 mg/kg/day. A 'No Observed Effect Level' (NOEL) has, therefore, not been achieved for males however the 'No Observed Effect Level' (NOEL) for females was, considered to be 15 mg/kg/day. The changes detected at 15 mg/kg/day were confined to minimal non-degenerative duodenum changes. In isolation and by that definition this was considered not to represent "serious damage" to health as defined by the criteria given in the EC labelling guide of Commission Directive 200l/59/EC. For this reason 15 mg/kg/day may be regarded as a "No Observed Adverse Effect Level" (NOAEL) for males.

Executive summary:

Introduction.The study was designed to investigate the systemic toxicity of the test material.It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).

Methods.The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to twenty-eight consecutive days, at dose levels of 15, 150 and 300 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

 

Mortality.One female treated with 300 mg/kg/day was found dead post dosing on Day 4. There were no other unscheduled deaths.

 

Clinical Observations.Animals of either sex treated with 300 mg/kg/day showed episodes of increased salivation around the time of dosing, noisy respiration and/or red/brown staining around the mouth/snout. One male treated with 150 mg/kg/day showed generalised fur loss and scab formation from Day 18 onwards and animals of either sex treated with 150 and 15 mg/kg/day showed incidents of increased salivation around the time of dosing during the final two weeks of treatment. The animal that died during the study showed no adverse clinical signs prior to death.

 

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

 

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

 

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

 

Bodyweight.Animals of either sex treated with 300 mg/kg/day showed a reduction in bodyweight gain during Week 4 only; statistical significance however was not achieved. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

 

Food Consumption.Animals of either sex treated with 300 mg/kg/day showed a reduction in food efficiency during Week 4. No such effects were detected in food efficiency for animals of either sex treated with 150 or 15 mg/kg/day or in food consumption for animals of either sex treated with 300, 150 or 15 mg/kg/day.

 

Water Consumption.No intergroup differences were detected.

Haematology.Animals of either sex treated with 300 mg/kg/day showed slight reductions in group mean haemoglobin, haematocrit and erythrocyte count. Associated changes for males also included a statistically significant reduction in mean cell haemoglobin concentration. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

 

Blood Chemistry.No toxicological significant changes were detected.

 

Organ Weights.No treatment-related effects were detected.

 

Necropsy.The female treated with 300 mg/kg/day that was found dead on Day 4 showed a dark liver and a raised limiting ridge in the stomach together with a pale and sloughing glandular gastric epithelium. One male and four females treated with 300 mg/kg/day together with two males and one female treated with 150 mg/kg/day showed a raised limiting ridge in the stomach. No such effects were detected in animals of either sex treated with 15 mg/kg/day.

 

Histopathology.The following treatment-related microscopic changes were detected:

 

BONE MARROW: A lower incidence of higher grades of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for females treated with 300 mg/kg/day. This was a marginal effect that was dependent upon three animals at the high dose level having lower grades of severity. There was no convincing effect at any other dose level, or among males at any dose level.

 

STOMACH:Gastric changes characterised by acanthosis/hyperkeratosis of the forestomach and limiting ridge, mucosal hypertrophy, and mucosal basophilia were seen among animals of either sex treated with 300 mg/kg/day or at 150 mg/kg/day, but not at 15 mg/kg/day.

 

DUODENUM:Mucosal hypertrophy was seen in relation to treatment for males only treated with 300 mg/kg/day, 150 mg/kg/day, and at 15 mg/kg/day with an apparent dose response in terms of severity grades. However there were no associated degenerative changes. Females were not similarly affected.

 

Conclusion.The oral administration of MAES to rats for a period of up to twenty-eight consecutive days at dose levels of 15, 150, and 300 mg/kg/day resulted in treatment-related effects in animals of either sex treated with 300 and 150 mg/kg/day and in males treated with 15 mg/kg/day. A 'No Observed Effect Level' (NOEL) has, therefore, not been achieved for males however the 'No Observed Effect Level' (NOEL) for females was, considered to be 15 mg/kg/day.

The changes detected at 15 mg/kg/day were confined to minimal non-degenerative duodenum changes. In isolation and by that definition this was considered not to represent "serious damage" to health as defined by the criteria given in the EC labelling guide of Commission Directive 200l/59/EC. For this reason 15 mg/kg/day may be regarded as a "No Observed Adverse Effect Level" (NOAEL) for males.

Cross-reference 3

Reason / purpose for cross-reference:

exposure-related information

Reference

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.

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

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)

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.

Remarks:

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

Remarks:

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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

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.

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

Reproductive effects observed:

not specified

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.

Data source

Reference

Reference Type:

other: Evaluation report

Title:

Unnamed

Year:

2019

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Evaluation of existing data from toxicity studies.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

57043-35-3

Cas Number:

57043-35-3

IUPAC Name:

57043-35-3

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

Specific details on test material used for the study:

See the respective data in the studies referred to in the evaluation report.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

other: See respective studies

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

Acute oral study; 28-day repeated dose study; screening study on reproduction and developmental toxicity.

Duration and frequency of treatment / exposure:

Up to eight weeks.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw (total dose)

Remarks:

Acute exposure.

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

Acute exposure

Doses / concentrations 3

Dose / conc.:

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

Remarks:

28-d repeated oral dose toxicity study/Reproduction-development screening study

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Reproduction-development screening study

Doses / concentrations 5

Dose / conc.:

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

Remarks:

28-d repeated oral dose toxicity study/Reproduction-development screening study

Doses / concentrations 6

Dose / conc.:

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

Remarks:

28-d repeated oral dose toxicity study

No. of animals per sex per dose / concentration:

See respective studies.

Positive control reference chemical:

Not applicable

Details on study design:

See respective study summaries

Details on dosing and sampling:

See respective study summaries

Statistics:

See respective study summaries

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Systemic effects were seen at 300 mg/kg bw/d secondary to the effects at the site of administration. Oral administration to rats at dose levels of 15, 50 and 150 mg/kg/day resulted only in loal effects associated with the irritating properties.

Main ADME results 2

Type:

distribution

Results:

Besides gastric irritation, there are no indications that the test item is distributed in such a way it affects other organs.

Main ADME results 3

Type:

metabolism

Results:

The products are likely to be a mixture of 2-acryloyloxyethanol, acrylic acid, mono-2-hydroxyethylhexahydrophthalic acid, hexahydrophthalic acid and ethane-1,2-diol (ethylene glycol).

Main ADME results 4

Type:

excretion

Results:

End products of metabolism are likely to include urinary metabolites and exhaled carbon dioxide.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The substance is both water soluble and fat soluble. Thus absorption following oral administration might be expected. It is possible that hydrolysis will take place before or during absorption, thus either the parent moiety or the hydrolysis products might be absorbed. Because of the corrosivity characteristics of the substance, absorption through the skin in the absence of hydrolysis is unlikely. Given the lack of vapour pressure, it is likely only little material is inhalable.

Details on distribution in tissues:

If absorbed and not metabolised, the substance is likely to partition between body water and fat (log Pow= 1.66). However, the substance will most likely be metabolised after uptake, thus accumulation in fat is unlikely.

Details on excretion:

Following oral administration, rt excreted unchanged, the substance is likely to appear in urine. The end products of metabolism are likely to include urinary metabolites and exhaled carbon dioxide.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Degradation of MAHP is caused by hydrolysis and metabolism. The products are likely to be a mixture of 2-acryloyloxyethanol, acrylic acid, mono-2-hydroxyethylhexahydrophthalic acid, hexahydrophthalic acid and ethane-1,2-diol (ethylene glycol). The hexahydrophthalic acid and 2-hydroxyethylhexahydrophthalic acid may be conjugated with glucuronic acid. The remainder are likely to enter intermediary metabolism and end products are likely to include significant amounts of carbon dioxide. Intermediary products may include the acid produced by oxidising the ethane-1,2-diol moiety.