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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 8th July 2008 and 10 March 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification : EA 2854
Description : Off white powder
Lot number : 1325J17001
Date received : 01 July 2008
Storage conditions : approximately 4°C in the dark

Test animals

Species:
rat
Strain:
other: Wistar Han™: HsdRccHan™: WIST
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Harlan Laboratories UK Ltd, Oxon, UK.

- Age at study initiation:
Parental animals - approximately six to eight weeks old
F1 generation - not applicable

- Weight at study initiation:
At the start of treatment the Subgroup A males weighed 263 to 305 g, the Subgroup A females weighed 140 to 199 g,
Subgroup B females were introduced to the study. Subgroup B females weighed 169 to 213 g

- Fasting period before study:
Not fasted

- Housing:
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).

- Diet (e.g. ad libitum):
A pelleted diet
(Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd, Oxon, UK) was used.

- Water (e.g. ad libitum):
The animals were allowed free access to food and water. Mains drinking water was supplied from polycarbonate bottles attached to the cage

- Acclimation period:
The animals were acclimatised for at least 6 days (Subgroup A) and 7 days (Subgroup B) during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21±2ºC

- Humidity (%):
55 ±15%

- Air changes (per hr):
The rate of air exchange was at least fifteen air changes per hour

- Photoperiod (hrs dark / hrs light):
low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: Day 1 To: Day 90

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the formulations to be stable for three hours. Formulations were therefore prepared daily. Test material formulations were analysed for concentration of EA 2854 at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.

For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals from both Subgroup A and Subgroup B were treated in an identical manner with 10 ml/kg/day of Arachis oil BP. For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material formulations were analysed for concentration of EA 2854 at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.
Details on mating procedure:
Subgroup A males were paired with Subgroup B females on a one male : one female basis within each dose group, for a period of up to twenty-one days. Once mating was confirmed, the male was paired with another female from the same dose group until mating was confirmed. This process was repeated until all females from each dose group were confirmed to have mated or twenty-one days had elapsed. 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 and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
90 days
Frequency of treatment:
For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals from both Subgroup A and Subgroup B were treated in an identical manner with 10 ml/kg/day of Arachis oil BP. For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Duration of test:
90 days
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Subgroup A:
10 male and 10 female

Subgroup B
0 Males and 22 Females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the formulations to be stable for three hours. Formulations were therefore prepared daily. Test material formulations were analysed for concentration of EA 2854 at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.

- Rationale for animal assignment:
Subgroup A animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure, and Subgroup B females were randomly allocated to tretament groups using random letter tables. The group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
For Subgroup B females, individual bodyweights were recorded on Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Day 0,7, 14 and 21 post coitum and on Days 1,4,7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
For Subgroup A females, food consumption was recorded for each cage group at weekly intervals throughout the study.

Dietary intake for Subgroup A males and Subgroup B females was recorded weekly for each cage group until pairing. Following confirmation of mating, dietary intake for Subgroup B females was recorded on Days ato 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4,4 to 7,7 to 14 and 14 to 21 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes / No / No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes

- Soft tissue examinations: Yes

- Skeletal examinations: Yes

- Head examinations: Yes
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance 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 Dennett’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.

The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value. Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥0.05 (not significant)

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (p) were calculated as follows:
p < 0.001 +++ --- ***
p < 0.01 ++ -- **
p < 0.05 + - *
p < 0.1 (+) (-) (*)
p ≥ 0.1 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is nondirectional.
Indices:
The following indices were calculated for each group from group mean data:
Live birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index 1(%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100
Viability Index 2 (%)= (Number of offspring alive on Day 7/Number of offspring alive on Day 4) x 100
Viability Index 3 (%) = (Number of offspring alive on Day 14/Number of offspring alive on Day 7) x100
Viability Index 4 (%) =(Number of offspring alive on Day 21/Number of offspring alive on Day 14) x100
Viability Index 5 (%) = (Number of offspring alive on Day 21/Number of offspring alive on Day 1) x100

ii) Sex Ratio (% males)
Group mean values calculated from each litter value on Days 1, 4, 7, 14 and 21 using the following formula:
(Number of male offspring/Total number of offspring) x100

iii) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated as follows:
% Pre-implantation loss = (Number of Corpora Lutea - Number of implantation sites/Number of corpora lutea) x100
% Post-implantation loss = [(Group number of implantation sites - Total number of offspring born)/Number of implantation sites] x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: No toxicologically significant effects were detected.

Details on maternal toxic effects:
With regards to reproduction, administration of the test material did not affect mating performance, fertility or reproductive performance in both Subgroup A males or Subgroup B females. There were no treatment-related effects detected in litter size or viability, growth or development of litters from treated animals and post-mortem findings of offspring did not reveal any treatment-related changes at the dose levels assessed in this study.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: No toxicologically significant effects were detected.

Details on embryotoxic / teratogenic effects:
With regards to reproduction, administration of the test material did not affect mating performance, fertility or reproductive performance in both Subgroup A males or Subgroup B females. There were no treatment-related effects detected in litter size or viability, growth or development of litters from treated animals and post-mortem findings of offspring did not reveal any treatment-related changes at the dose levels assessed in this study.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment related effects were seen, therefore the NOAEL has been determined to be 1000 mg/kg bw/day for both systemic and reproductive toxicity.
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of EA 2854 by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.
Executive summary:

Introduction. The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983). The study was also designed to assess subchronic exposure to the test material to the rat and is based on the OECD Guidelines for Testing of Chemicals No 408 "Subchronic Oral Toxicity - Rodent" (Adopted 21 September 1998).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 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. For the Subchronic phase (Subgroup A), the test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™: HsdRccHan™: WIST strain rats, for one hundred and eight consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, bodyweight development, dietary intake and water consumption were monitored during the study. Haematology, blood chemistry and urinalytical investigations were undertaken for all Subgroup A animals during Week 10. Ophthalmoscopic examination was also performed on Subgroup A control and high dose animals once prior to the start of treatment and during Week 10. During Week 11, sensory reactivity, grip strength and motor activity assessments were performed.

During Week 8, females for the reproductive phase (Subgroup B) were introduced to the study. The test material was administered to gavage to three groups, each of twenty-two female Wistar HanTM: HsdRccHanTM: WIST strain rats,for at least twenty-eight days prior to mating and throughout the mating, gestation and lactation phases of the study at dose levels of 50, 250 and 1000 mg/kg/day. A control group of twenty-two females were dosed with vehicle alone (Arachis oil BP).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored for Subgroup B females. Oestrus cycle assessments were also undertaken from the start of treatment until pairing.

After Week 11 of the study, Subgroup A males were paired with Subgroup B females on a one male: one female basis within each dose group, for a maximum of twenty-one days. Pairing was continued until all twenty-two females from each group had mated with a Subgroup A male from the same dose group. On completion of the mating phase, all Subgroup A animals, were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

All pregnant Subgroup B females were allowed to girth birth and maintain their offspring until Day 21 post parium. Offspring development was observed during this time. On Day 21 post parium, females and their offspring were subjected to a gross necropsy examination and histopathological examinations of reproductive tissues from high dose and control females was performed.

Results.

Mortality: There were no unscheduled deaths during the study in both Subgroup A or Subgroup B.

Clinical observations: No clinical:y observable signs of toxicity were detected throughout the treatment period for both Subgroup A and Subgroup B.

Behavioural assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Functional performance tests: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Sensory reactivity assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Bodyweight: No adverse effect on bodyweight development was detected for Subgroup A animals throughout the treatment period, or for Subgroup B females prior to mating, or during the gestation and lactation phases of the study.

Food consumption: No adverse effects on dietary intake or food efficiency were detected for Subgroup A animals during the treatment period, or for Subgroup B females during the pre-mating, gestation and lactation phases of the study.

Water consumption: No intergroup differences were detected for treated animals from both Subgroup A and Subgroup B when compared to their concurrent controls.

Ophthalmoscopy:No ocular effects were detected for high dose Subgroup A animals.

Urinalysis: There were no treatment-related effects detected in the urinalytical parameters investigated for Subgroup A animals.

Haematology:No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Blood chemistry: No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Oestrus cycle assessments: No significant differences in oestrus cycles were detected for treated Subgroup B females when compared to their concurrent controls.

Mating performance: There were no treatment-related differences in mating performance.

Fertility & pregnancy: There were no treatment-related effects detected on fertility or pregnancy status for treated animals when compared to controls.

Gestation length: No treatment-related effects on gestation lengths were detected for treated Subgroup B females when compared to controls.

Litter responses: There were no treatment-related effects detected for litters from treated animals when compared to controls. There were no differences in corpora lutea or pre/post implantation losses for treated animals when compared to controls.

No treatment-related effects were detected for offspring during the daily observations, litter weights or offspring bodyweights. Reflexological assessments did not reveal any significant differences between litters from treated and control dams. No treatmentrelated macroscopic abnormalities were detected at termination.

Semen assessment: No treatment-related effects were detected in sperm motility, morphology or homogenisation resistant spermatid counts from treated Subgroup A males when compared to concurrent controls.

Organ weights: No toxicologically significant intergroup differences were detected for both Subgroup A animals and Subgroup B females.

Necropsy: No treatment-related macroscopic abnormalities were detected for both Subgroup A and Subgroup B animals at terminal kill.

Histopathology: No treatment-related microscopic abnormalities were detected for animals from both Subgroup A and Subgroup B.

Conclusion: Oral administration of EA 2854 by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.