Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
Pre-natal developmental toxicity study.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 14 September 2016 and experimental completion date: 19 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’
Version / remarks:
Adopted 22 January 2001
Deviations:
yes
Remarks:
Please see "Any other information on materials and methods incl. tables" field below.
Qualifier:
according to
Guideline:
other: US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’
Version / remarks:
(August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: 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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
Not applicable.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
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, Envigo RMS (UK) Limited, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Annex 2. 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 Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility.

Animal Information
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 181 to 299 g.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 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.
Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records

IN-LIFE DATES: From: To: The in-life phase of the study was conducted between 16 September 2016 (first day of treatment) and 05 October 2016 (final day of necropsy).


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
Remarks on MMAD:
Not applicable
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Test Item Preparation and Analysis
For the purpose of the 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 determined during Envigo Study Number: TS44LC by Envigo Research Limited, Shardlow, UK Analytical Services. Results showed the formulations to be stable for at least twelve days at 2.5 mg/mL and 21 days at 250 mg/mL.
Formulations were therefore prepared weekly and stored at approximately +4 °C in the dark.

Samples were taken of test item formulation and were analyzed twice for concentration of Araldite CY 184 at Envigo Analytical Laboratory, Shardlow.
The results indicate that the prepared formulations were within ± 10% of the nominal concentration confirming accurate formulation.
Details on mating procedure:
Female were already mated when the test item was administred therefore no information on matting procedure is available.
Animals were delivered in 2 batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
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 high performance liquid chromatograhy - mass spectrometry (HPLC-MS) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test item supporting informations: the test item described in the main part of this study was also used as the analytical standard.

Apparatus: Analytical and top-pan balances.


Reagents:
- Control vehicle: PEG 400
- Acetonitrile: Gradient HPLC grade
- Water: LC-MS
- Formic acid: Analytical reagent
- Dilution solvent: Acetonitrile

Preparation of calibration standards:
Stock solutions of the test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.05 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration range of 0.001 mg/mL to 0.025 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum.

Preparation of Test Samples
The formulations received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.
Instrumentation Parameters
HPLC System : Agilent Technologies 1200 MSD, incorporating autosampler and workstation
Mass selective detector
Source: electrospray
Fragmentation energy: 110volts
Polarity: positive
Mode: single ion mode with 302.1 amu
Gas temperature: 350 °C
Drying gas: 9 litre/minute
Nebuliser pressure: 40 psi
Capillary voltage: 1500 volts
Gain: 1
Column: Symmetry, 3.5 microm, (50 x 3 mm id)
Column temperature: 30 °C
Gradient elution: eluent A: 10 mM ammonium formate with 0.1% formic acid in LC-MS water eluent B: 0.1% formic acid in methanol

Time (mins) %A %B
0 95 5
5 5 95
10 5 95

- Flow rate: 0.4 mL/min
- Injection volume: 5 microL
- Retention time: approximately 6.8 minutes


Data Evaluation and Calculations

The peak area response for Araldite CY 184 in each calibration standard chromatogram was measured. Calibration curves were constructed by non-linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved

The recovery rate (R) of the spiked recovery sample was calculated using the following equation:

R= (C / C fort)x100

where R: recovery rate [%]
c: determined concentration of the test item in the spiked recovery sample [mg/mL]
cfort: fortified concentration of the test item in the spiked recovery sample [mg/mL]

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

Concentration of Dose Formulations
The mean concentrations were within applied limits ±10%, confirming accurate formulation.

CONCLUSION
The mean concentrations of Test Item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
The test item was administred daily, from day 3 to day 19 of gestation by gavage.
Control animals were treated in an identical manner with the vehicle alone.
Frequency of treatment:
Daily
Details on study schedule:
Not Applicale as the study is a Pre-natal developmental (OECD 414).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
Dose / conc.:
175 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Treatment volume: 4 mL/kg
No. of animals per sex per dose:
24 animals mated females rats.
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: 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.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
MATERNAL OBSERVATIONS AND EXAMINATIONS:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Day 20 of gestation
All surviving 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.
Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
No
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter data, Litter placenta, fetal weight, fetal examination,

GROSS EXAMINATION OF DEAD PUPS:
No dead Pups.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
All surviving 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. 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 as follows (as an example):

Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus
Postmortem examinations (offspring):
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. With the exception of four fetuses (V8, V10, V12 and V14) from litter 30 which were not identified#, alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. 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 into 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, on 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 nonparametric 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)
Reproductive indices:
N/A
Offspring viability indices:
N/A

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
At 350 mg/kg bw/day, intermittent instances of increased salivation (generally post-dosing only) were observed in most females between Days 11 and 19 of gestation with 3/24 females from the 175 mg/kg bw/day dose group also exhibiting these observations between Days 11 and 17 of gestation. Such findings are common in this type of study and usually reflect unpalatability and/or an irritant nature of the test item formulation and are generally deemed to be of no toxicological importance. A few instances of noisy respiration were also noted in surviving females from these dose groups (2/23 and 3/24 females treated with 175 or
350 mg/kg bw/day, respectively) during the latter half of the dose administration period. This observation was also deemed to be the result of test item irritancy and/or gavage-related reflux rather than an indication of the systemic toxicity of the test item.

One female treated with 175 mg/kg bw/day showed generalized fur loss between Days 16 and 17 of gestation with one female each from the 50 mg/kg bw/day and control dose groups also showing isolated instances of noisy respiration. These findings were considered likely to be incidental.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female 52 treated with 175 mg/kg bw/day was killed on Day 14 of gestation (before dosing) in animal welfare grounds due to a decline in animal condition. Prior to death, clinical signs for this female included gasping respiration, decreased respiratory rate, hunched posture, piloerection, ptosis, lethargy and chromodacryorrhea, with noisy respiration noted on Day 13 of gestation. At necropsy, macroscopic findings were confined to enlarged thymus and fluidfilled thoracic cavity indicating dosing trauma to be the main cause of death.
There were no further unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the start of dosing (over Day 3 to 4 of gestation), group mean body weight gain in females treated with 350 mg/kg bw/day was statistically significantly lower than controls (p<0.001). Subsequent improvement was evident but the mean body weight gains in these females remained lower than controls up to Day 11 of gestation with statistical significance attained up to Day 8 of gestation (p<0.05). Towards the end of the dose administration period (over Days 17 to 20 of gestation), group mean body weight gain in these females was again statistically significantly lower than controls (p<0.05). Due to these effects on body weight development, cumulative body weight gains in these females remained statistically significantly lower than controls throughout the dosing period (p<0.001) with the intergroup difference in group mean body weight from controls on Day 20 of gestation also achieving statistical significance (p<0.05). There was no effect of treatment with the test item at this dose level on gravid uterus weight, but group mean body weight on Day 20 of gestation and overall body weight gain, when adjusted for the contribution of gravid uterus weight, were statistically significantly lower than controls (p<0.05 and p<0.001, respectively).

At 175 or 50 mg/kg bw/day, there was no effect of treatment on body weight development throughout the dosing period. Group mean gravid uterus weight in these females was also similar to controls together with the mean body weights and mean body weight gains, when adjusted for the contribution of gravid uterus weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 350 mg/kg bw/day, group mean food consumption remained slightly lower than controls between Days 3 and 14 of gestation with statistical significance achieved up to Day 11 of gestation (p<0.01-p<0.001). Towards the end of the dose administration period (over Days 17 to 20 of gestation), dietary intake in these females was again statistically significantly lower than controls (p<0.05).
At 175 or 50 mg/kg bw/day, there was no effect of treatment on food intake throughout the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the scheduled terminal necropsy on Day 20 of gestation, treatment-related macroscopic findings included 4/24 females treated with 350 mg/kg bw/day showing raised limiting ridge in the stomach with another female from this dose group exhibiting sloughing and raised white patches in the non-glandular region of the stomach.
Other macroscopic findings were confined to one female each from the 350 or 50 mg/kg bw/day dose groups showing lungs with dark patches; lungs from this 350 mg/kg bw/day female also appeared to be pale. A dose-relationship was not evident and due to the isolated nature of these observations, they were deemed unlikely to be related to treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The following assessment is based on the 24, 23, 23 and 24 females with live young on Day 20 of gestation at 0 (control), 50, 175 and 350 mg/kg bw/day, respectively. Female 42 from the 50 mg/kg bw/day dose group was found not to be pregnant. This was an isolated event considered to be unrelated to treatment with the test item. The early decedent (Female 52 treated with 175 mg/kg bw/day) was confirmed to be pregnant (at necropsy).
There was no effect of maternal treatment on litter data as assessed by the mean number of implantations, in-utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size and post-implantation losses at 50, 175 or 350 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Pre-implantation losses and sex ratios across all test item-treated dose groups were also similar to controls. Additionally, fetal, litter or placental weights remained unaffected by treatment with the test item at all dose levels.

FETAL EXAMINATIONS:
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities. A small number of fetuses from the 175 mg/kg bw/day dose group were noted with enlarged placentae; however, most of these fetuses were from one litter and in the absence of similar findings in the 350 mg/kg bw/day dose group, this observation was considered unlikely to be treatment-related.

A statistical analysis of the skeletal evaluation data identified a statistically significant increase in the incidence of ossification of one or more forepaw phalanges in offspring from Group 4 when compared to controls (p<0.05). Although the group mean value was outside the historical control data range, in the absence of a true dose-related response and due to the isolated nature of this observation, it was considered unlikely to represent an adverse effect of treatment with the test item on fetal development. Any other minor intergroup differences did not attain statistical significance and were generally not dose-dependent and, as such were considered likely to be due to biological variation.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
350 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Please refer to the discussion part.

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

DISCUSSION:

Treatment of pregnant female rats with Araldite CY 184 at a dose level of 350 mg/kg bw/day resulted in a reduction in periodic body weight gains over Days 3 to 11 and 17 to 20 of gestation. This caused lower cumulative body weight gains for these females throughout the dosing period and an approximately 16% reduction in overall body weight gain. The effect on body weight development was associated with a slight reduction in dietary intake over most of the dosing period, which appeared to be more prominent at the start of the treatment.

Group mean body weight and overall body weight gain, when adjusted for the contribution of gravid uterus weight, were also lower than controls. Similar effects were not evident in females treated with 175 or 50 mg/kg bw/day.

In isolation, the effects on body weight performance and dietary intake as well as macroscopic necropsy observations in pregnant rats given 350 mg/kg bw/day were considered to be of an adverse nature. It is worth noting, however, that such observations are often suggestive of an irritant effect of the test item rather than an indication of its systemic toxicity. Such irritancy was previously observed with this test item in a ninety day repeated dose oral (gavage) toxicity study in the rat (Envigo Study Number TS44LC) whereby histopathological examination of the stomach from females treated with 350 mg/kg bw/day

identified microscopic changes consistent with an irritant effect of the test item. These microscopic alterations, confined to an area of the stomach which is unique to the rodent anatomy and therefore deemed to be of little significance in man, correlated with macroscopic observations noted in the stomach; similar macroscopic findings at necropsy were observed in some females in the present study. Intermittent instances of increased salivation observed in females treated with 350 or 175 mg/kg bw/day during the treatment period were also considered to reflect unpalatability and/or an irritant nature of the test item

formulations with isolated episodes of noisy respiration in some of these females also likely due to the irritant nature of the test item formulation and/or gavage-related reflux.

Treatment up to a dose level of 350 mg/kg bw/day did not result in any treatment-related effects on fetal survival or weights. At 350 mg/kg bw/day, a statistically significant increase in the incidence of ossification of one or more forepaw phalanges was noted. This observation is a minor variant and although the group mean value was outside the historical control data range, due to the isolated nature of this finding and the lack of a true-dose relationship, it was deemed not to represent an adverse effect of treatment with the test item.

A true ‘No Observed Adverse Effect Level’ (NOAEL) for maternal toxicity in the pregnant rat in this study was considered to be 175 mg/kg bw/day. Additionally, the NOAEL for developmental toxicity was considered to be 350 mg/kg bw/day within the confines of this study.

CONCLUSION

The oral administration of Araldite CY 184 to pregnant rats by gavage during gestation Days 3 to 19, at dose levels of 50, 175 or 350 mg/kg bw/day resulted in adverse effects on body weight gain and food consumption at the high dose level. Although some improvement was evident after the initial effects, cumulative body weight gain for females treated with 350 mg/kg bw/day remained lower than controls throughout the dosing period with the overall body weight and body weight gain adjusted for the contribution of gravid uterus weight also being lower than controls. At necropsy, a small number of these females showed macroscopic findings in the stomach. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the maternal toxicity in the pregnant rat was

considered to be 175 mg/kg bw/day within the confines of this type of study.

At 350 mg/kg bw/day, skeletal examination identified an increase in the ossification of one or more forepaw phalanges. As this observation is only a minor variant and there were no other treatment-related effects on fetal development, 350 mg/kg bw/day was considered to be the NOAEL for developmental toxicity within the confines of this study.

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance to pregnant rats by gavage during gestation Days 3 to 19, at dose levels of 50, 175 or 350 mg/kg bw/day resulted in adverse effects on body weight gain and food consumption at the high dose level. Although some improvement was evident after the initial effects, cumulative body weight gain for females treated with 350 mg/kg bw/day remained lower than controls throughout the dosing period with the overall body weight and body weight gain adjusted for the contribution of gravid uterus weight also being lower than controls. At necropsy, a small number of these females showed macroscopic findings in the stomach. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the maternal toxicity in the pregnant rat was considered to be 175 mg/kg bw/day within the confines of this type of study.

At 350 mg/kg bw/day, skeletal examination identified an increase in the ossification of one or more forepaw phalanges. As this observation is only a minor variant and there were no other treatment-related effects on fetal development, 350 mg/kg bw/day was considered to be the NOAEL for developmental toxicity within the confines of this study.
Executive summary:

The study was performed 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 3 and 19 of gestation, inclusive, at dose levels 50, 175, or 350 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Polyethylene Glycol 400) to serve as a control.

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

All surviving 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 treatment-related deaths during the study.

Female 52 treated with 175 mg/kg bw/day was killed on Day 14 of gestation (before dosing) on animal welfare grounds due to a decline in condition. This was considered to be due to a dosing trauma.

Clinical Observations

There were no clinical observations of toxicological significance at any dose level.

Body Weight

At 350 mg/kg bw/day, group mean body weight gains were lower than controls between Days 3 and 11 and Days 17 and 20 of gestation. Some improvement was observed after the initial effect, but cumulative body weight gains remained lower than control throughout the treatment period, with the overall body weight gain in these females approximately 16% lower than controls. Group mean body weight and overall body weight gain, when adjusted for the contribution of gravid uterus, were also lower than controls. These differences were considered to be due to an irritant effect of the test item.

At 175 or 50 mg/kg bw/day, there was no effect of treatment with test item on body weight development.

Food Consumption

At 350 mg/kg bw/day, dietary intake remained lower than control through most of the dosing period with the effect appearing to be more prominent at the start of dose administration.

This decrease in food consumption was considered to be due to an irritant effect of the test item.

At 175 or 50 mg/kg bw/day, there was no effect of treatment with test item on food intake.

Water Consumption

Visual inspection of water bottles did not indicate any intergroup differences in water consumption levels for test item-treated females when compared with controls.

Post Mortem Studies

Treatment-related findings were confined to 4/24 females treated with 350 mg/kg bw/day

showing raised limiting ridge in the stomach with another female from this dose group

exhibiting sloughing and raised white patches in the non-glandular region of the stomach.

These findings were considered likely to be due to an irritant effect of the test item.

Litter Data and Litter Placental and Fetal Weights

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

Fetal Examination

No adverse effects were detected during external examination of the fetuses or in the type and incidence of skeletal or visceral findings.

Conclusion

The oral administration of Araldite CY 184 to pregnant rats by gavage during gestation Days 3 to 19, at dose levels of 50, 175 or 350 mg/kg bw/day resulted in adverse effects on body weight gain and food consumption at the high dose level. Although some improvement was evident after the initial effects, cumulative body weight gain for females treated with 350 mg/kg bw/day remained lower than controls throughout the dosing period with the overall body weight and body weight gain adjusted for the contribution of gravid uterus weight also being lower than controls. At necropsy, a small number of these females showed macroscopic findings in the stomach. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the maternal toxicity in the pregnant rat was considered to be 175 mg/kg bw/day within the confines of this type of study.

At 350 mg/kg bw/day, skeletal examination identified an increase in the ossification of one or more forepaw phalanges. As this observation is only a minor variant and there were no other treatment-related effects on fetal development, 350 mg/kg bw/day was considered to be the NOAEL for developmental toxicity within the confines of this study.