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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
Study period:
27 March 2014 - 10 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Monitoring authority, Dept. of Health UK, 2014
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): AS1100
- Description: Amber colored liquid
- Purity: 98%
- Batch Number: 5500020562
- Date Received: 15 January 2014
- Storage Conditions: Ambient temperature 10 °C to 30 °C in the dark
- No correction for purity was made.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 196 to 269g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 14. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Test item formulation and experimental preparation
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Arachis Oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services as part of Harlan Laboratories Ltd. Study Number: 41304264 and results showed the formulations to be stable for twenty-one days. Formulations were therefore prepared once and stored at approximately +4 °C in the dark.

Samples were taken of each test item formulation and were analyzed for concentration of AS1100 at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.
The formulations investigated during the study were found t ocomprise test item in the range of 101 to 110 % and thus ther required content limit with reference to the nominal concentration was met.
The test item was found to be stable in the formulations when kept for 21 days in the refrigerator ( 4°C).
In conclusion, the restults indicated the accurate use of the test item and vehicle during this study, The formulations were found t obe homogenously prepared and sufficient formulation stability under storage conditions was proven.
Details on mating procedure:
Not specified
Duration of treatment / exposure:
The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage.
Frequency of treatment:
Daily
Duration of test:
The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24 females / group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen based on previous toxicity data.

Examinations

Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position
Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative (excluding fetuses in the litter of Female No. 83). Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, an alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths.


Clinical Observations

Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Eighteen females treated with 1000 mg/kg bw/day showed episodes of increased salivation from Day 12 onwards. One female treated with 300 mg/kg bw/day had increased salivation from Day 18 onwards. Observations of this nature are commonly experienced following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.

No such effects were detected in females treated with 100 mg/kg bw/day.

Body Weight
No treatment-related effects in body weight development were detected.
Statistical analysis of the data did not reveal any significant intergroup differences.

Food Consumption
No treatment-related effects were detected in food consumption.
Statistical analysis of the data did not reveal any significant intergroup differences.

Water Consumption
Daily visual inspection of water bottles did not reveal any overt intergroup differences.


Post Mortem Studies
No macroscopic abnormalities were detected.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other:
Remarks:
No toxicologically significant effects were noted in parental females.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Data and Litter Placental and Fetal Weights
There were no obvious adverse effects of maternal treatment on litter data as assessed by the number of implantations, early and late embryonic/fetal deaths and live fetuses or sex ratio, as assessed by percentage male.

Females treated with 300 mg/kg bw/day had a statistically significant increase in the number of corpora lutea. In the absence of a true dose related response, this intergroup difference was considered to represent normal biological variation rather than an effect of treatment.

Fetal Examination
For all dose groups, there were no significant treatment-related trends in the proportion of fetuses (or litters) with evidence of visceral or skeletal anomalies. The type of visceral and skeletal anomalies were those commonly observed for this type of study.

Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in the number of fetuses with dumb-bell shaped thoracic centrum. The lower incidence of this parameter was considered to indicate a higher number of fetuses showing normal development of the thoracic centrum. Therefore, in isolation and in the absence of any differences in a number of variants or a syndrome of variance, the intergroup difference was considered not to be toxicologically significant.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in the number of fetuses showing incomplete ossification of the thoracic centrum. In the absence of a true dose relationship and in isolation the intergroup difference was considered to be of no toxicological importance.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicological significant changes were detected in the offspring parameters measured.
Remarks on result:
other: Max. dose tested

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of AS1100 to pregnant rats by gavage during gestation at dose levels of 100, 300, 1000 mg/kg/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

Introduction

The study was performed according to the study plan presented in Appendix13and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

 

The study was designed to comply with the following guidelines:

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (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 twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis Oil BP) to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results…….

Mortality

There were no unscheduled deaths.

Clinical Observations

Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Body Weight

No treatment-related effects in body weight development were detected.

 

Food Consumption

No treatment-related effects were detected in food consumption.

 

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

 

Post Mortem Studies

No macroscopic abnormalities were detected.

 

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

 

Fetal Examination

No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

 

Conclusion

The oral administration of AS1100 to pregnant rats by gavage during gestation at dose levels of 100, 300, 1000 mg/kg/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.