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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD Guideline 421 under GLP.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was considered as reliable without restriction and performed under GLP.
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

Oral (Gavage) Reproduction/Devlopmental Toxicity Screening Test in the Rat

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.

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 two 150-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.

Short description of key information:

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. However, none of the dosages applied had any adverse impact on reproductive performance.

Justification for selection of Effect on fertility via oral route:

Compatible with OECD Guidelines for Testing of Chemicals No. 421.

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. However, none of the dosages applied had any adverse impact on litter size, viability, sex ratio, body weights behaviour or physical development.

Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was considered as reliable without restriction and performed under GLP.
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

Oral (Gavage) Reproduction/Devlopmental Toxicity Screening Test in the Rat

Results

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.

Justification for selection of Effect on developmental toxicity: via oral route:

Compatible with OECD Guidelines for Testing of Chemicals No. 421.

The submission substance is not considered classified with regards to reproductive toxicity.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 20/07/2010 Date of signature: 29/10/2010
Limit test:
no

Test material

Constituent 1
Chemical structure
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

Administration / exposure

Route of administration:
oral: gavage
Details on mating procedure:
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.
Duration of treatment / exposure:
Eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Daily
Duration of test:
Eight weeks
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Concentrations: 3.75 mg/ml
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Concentrations:12.5 mg/ml
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Concentrations: 37.5 mg/ml
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
GENERAL
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.

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.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Yes
- Time schedule:
- Immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, 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 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. 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 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.

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

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
Ovaries and uterine content:
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
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)
vi) landmark developmental signs
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)
Quantitative data were analysed by the Provantis TM Tables and Statistics Module. After appropriate transformation, the use of possible covariates was 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 tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskai-Wallis ANOVA and Mann-Whitney 'U' test. Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (P) were calculated as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
p>=0.05 (not significant)
Indices:
Gestation Length, Parturition Index, Implantation Losses (%), Live Birth and Viability Indices, Sex Ratio (% males)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
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.
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Statistical analysis did not reveal any significant intergroup differences.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to slight (grade 1-2) hyperplasia/hyperkeratosis in the forestomach (nonglandular part) and erosion/sloughing, vascular dilatation and mixed cell infiltration were seen in the glandular part of the stomach in 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. These findings were seen at a higher incidence in the males than in the females and corresponded to the macroscopic findings seen at necropsy, raised limiting ridge, thickening of the glandular region and red patches in the glandular region and were considered to be a direct effect of the locally irritant properties of the test item on the mucosa of the stomach.
No treatment related findings were detected in the reproductive organs and the few other findings are those which are commonly found in the untreated laboratory rat of this strain and age and were considered to be of no toxicological importance.
Histopathological findings: neoplastic:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
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.
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
< 15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No significant differences in litter size, viability or sex ratio were evident for offspring from treated litters when compared to those from the controls.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Post natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

The oral administration of MAES to rats for a period of up to forty-two days for males and up to eight weeks for females (including two weeks pre-mating, gestation and early lactation period) at dose levels of 15, 50 and 150 mg/kg/day did not result in reproductive effects at the dose levels examined. There were no effects on mating performance or fertility. Furthermore, there was no evidence of any developmental effects observed in offspring from treated litters and no effect on the reproductive organs was evident following post mortem assessments or during histopathological assessments. Therefore, a clear 'No Observed Effect Level' for

reproductive toxicity was established at 150 mg/kg/day.

Although there were no treatment-related effects that affected reproductive performance the following findings were evident:

- Clinical signs of increased salivation developed in 150 mg/kg/day males and females from Day 2 onwards with similar findings evident intermittently in males treated with 50 mg/kg/day from the second week of treatment. Isolated instances of staining around the mouth, noisy respiration and sneezing were also observed in test animals during the study. These observations are commonly observed following the oral administration of

an unpalatable or slightly irritant test item formulation.

- Further evidence of a response to oral gavage administration of a slightly irritant test item was evidenced by slightly low body weight gains (when compared to controls) in 150 mg/kg/day males during the second week of treatment and post mortem findings of gastric inflammation identified as a raised limiting ridge and thickening of the glandular gastric epithelium 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 two 150 mg/kg/day males showed reddened patches. These macroscopic findings

corresponded with histopathological changes involving minimal to slight severities of hyperplasia/hyperkeratosis in the forestomach (non-glandular part) and erosion/sloughing, vascular dilatation and mixed cell infiltration in the glandular part of the stomach in 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.

- Similar locally irritant properties of the test item were evident in the previous twenty-eight day study (Study No. 2251-0026). Based on results from this study such changes were indicated to have no impact in the reproductive performance of test animals.

Applicant's summary and conclusion

Conclusions:
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. However, none of the dosages applied had any adverse impact on litter size, viability, sex ratio, body weights behaviour or physical development.