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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:
27/04/2013 - 07/06/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-chloroethoxy)methane
EC Number:
203-920-2
EC Name:
Bis(2-chloroethoxy)methane
Cas Number:
111-91-1
Molecular formula:
C5H10Cl2O2
IUPAC Name:
1-chloro-2-[(2-chloroethoxy)methoxy]ethane
Details on test material:
Substance name: Bis-(2-chloroethoxy)methane
Batch number: 99016
Appearance: Slightly yellowish liquid
Purity: 81.4%

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: S&K-Lap Kft., 2173 Kartal, Császár út 135, Hungary
- Age at study initiation: Young adult female rabbits, nulliparous and non-pregnant, approximately 4-5 months old
- Weight at study initiation: -
- Fasting period before study: -
- Housing: Rabbits will be individually housed in AAALAC approved metal wire rabbit cages (65 x 65 cm with height of 45 cm, on 2 levels with a shelf to allow rabbits to climb up to stretch out). Cages are of an open wire structure and are placed together to allow some social interaction with rabbit(s) in adjoining cages. Toys may be offered to the animals for enrichment purposes.
- Diet (e.g. ad libitum): Animals will be provided with UNI diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.
- Water (e.g. ad libitum): tap water as for human consumption, ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: 27/04/2013 - 07/06/2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): most suitable after trial formulations
- Concentration in vehicle: 0, 6, 20 or 60 mg/ml
- Amount of vehicle (if gavage): 0.5 ml/kg
- Lot/batch no. (if required):
Name: Polyethylene glycol 400
Lot No.: BCBJ7623V*
Manufacturer: Sigma-Aldrich
* Other lots may be used as required; to be documented in the raw data and reported.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All formulations were found to be in the range of 96 to 102% of nominal concentrations.
Details on mating procedure:
- Impregnation procedure: artificial insemination
Synchronisation of the oestrus cycle of the does will be performed 48 hours prior to insemination by administration of PMSG (gonadotropin) hormone (40 IU/female, sc). The female rabbits will be artificially inseminated. The insemination procedure will be performed in batches (see Appendix 2 for the tentative schedule) by the breeder at CiToxLAB Hungary Ltd., with sperm originated from New Zealand White male rabbits from the same source as the females. Each female will be inseminated with diluted sperm containing at least 2 million spermatozoa. At the same time as the artificial insemination is performed, ovulation will be stimulated with 1 ml buserelin-based compound (0.2 mL/animal, i.m.) (see Details of Other Materials section). The day of insemination is regarded as Gestation Day 0 (GD0).
Duration of treatment / exposure:
gd 6-27
Frequency of treatment:
daily
Duration of test:
28 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 3, 10, 30 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set by the Sponsor in consultation with the Study Director based on available data, including results of two preliminary studies [CiToxLAB study code 12/094-028N, “Bis-(2-chloroethoxy)methane: A Maximum Tolerated Dose and 21-Day Toxicity Study Following Oral (Gavage) Administration in New Zealand Rabbits” and 12/094-105NE, “Bis-(2-chloroethoxy)methane: A Dose Range Finding Toxicity Study Following Oral (Gavage) Administration in Pregnant New Zealand Rabbits”], with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for animal assignment (if not random): The inseminated, assumed pregnant female rabbits will be allocated to the dose groups on each insemination day in such a way that the group averages of the body weight will be as similar as possible.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- Animals will be inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Animals that show severe clinical signs will be isolated and may be sacrificed to prevent suffering and/or autolysis, and will be processed in the same way as the animals of the terminal necropsy.
As of GD0, clinical observations will be made at least daily when the peak of the clinical observations, if any, is observed after treatment, or as practical (but at least once) during the working day, on the days with no dose administration. When signs of toxicity are noted, animals may be observed more frequently.
Detailed clinical observations will be made on all animals at the onset of treatment (GD6) then at least weekly.
Pertinent behavioural changes and all signs of toxicity including mortality will be recorded including onset, degree and duration of signs as applicable. Signs evaluated will include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) will also be recorded. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal will be recorded with precision of 1 g on the day of insemination, then at least on the first day of dosing, every three days and on the day of scheduled necropsy, e.g. GD0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The food will be measured with precision of 1 g at approximately 3-day intervals, ex. on GD0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Food consumption will be calculated for each interval, including GD 0-28.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #28
- Organs examined: The doe’ viscera will be examined macroscopically for any structural abnormalities or pathological changes; the stomach and the reproductive organs from all animals and all the gross findings will be retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, in any abnormalities noted) for possible future evaluation.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The statistical evaluation of all numerical data will be performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method, including Bartlett, ANOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, Chi2, and/or exact Fisher test, as applicable.
The homogeneity of variance between groups will be checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) will be made. If the obtained result is significant Duncan’s Multiple Range test will be used to assess the significance of inter-group differences. Significant results with inter-group comparisons will be further compared using Kruskal-Wallis, and Mann-Whitney U-tests.
The limit for growth retarded foetuses will be calculated from the average body weight of the vehicle control foetuses. A foetus is considered as growth retarded if the deviation from the mean control values is greater than minus two fold standard deviation of all control foetuses.
Non pregnant females, females with no implantation, or ≤ 3 implantation sites, and females showing signs of abnormal pathology and/or misdosing will be excluded from statistical analysis; in-life individual data will be reported as applicable.
Indices:
Maternal Data:
- Clinical signs (by gestation day)
- Mortality (by gestation day), if any
- Body weight and body weight gain: mean ± SD
- Corrected body weight on GD28 (body weight-gravid uterine weight) and corrected body weight gain: mean ± SD
- Food consumption: mean ± SD
- Pathology findings
- Gravid uterine weight

Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± SD
- Number of implantations: mean ± SD
- Number and percent of live foetuses: mean ± SD
- Number and percent of intrauterine mortality: mean ± SD
Classified according to time of death: Pre-implantation loss, Post-implantation mortality, Early and late embryonic, as well as foetal death
o Pre-implantation loss: %, group mean
Number of corpora lutea-Number of implantations/Number of corpora lutea x100
o Post-implantation loss: %, group mean
Number of implantations-Number of live foetuses/Number of implantations x100

Foetal Data:
- Sex distribution: %, group mean
Number of male (female) foetuses/Number of foetuses x100
- Foetal body weight (accuracy 0.1 g): mean ± SD
- Crown-rump length (accuracy 1 mm): mean ± SD
- External abnormalities/litter: %, group mean
Number of foetuses with external abnormality/Number of foetuses x100
- Visceral abnormalities/litter: %, group mean
Number of foetuses with visceral abnormality/Number of foetuses x100
- Skeletal abnormalities/litter: %, group mean
Number of foetuses with skeletal abnormality/Number of foetuses x100
Historical control data:
Yes, data is included in the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: , fetotoxicity was observed.

Details on maternal toxic effects:
No clinical signs or mortality considered related to Bis-(2-chloroethoxy)methane were noted during the study following administration to artificially-inseminated females at dose levels up to and including 30 mg/kg bw/day. In the control, low, mid and high dose groups there were 0/24, 5/24, 1/24 and 2/24 rabbits found dead or euthanized, respectively. The early deaths were not related to maternal toxicity with test item. Three deaths were due to spontaneous abortions, they were euthanized. The other deaths were attributed to misgavage, reflux/aspiration of the vehicle and/or possible subsequent inflammatory/pulmonary infection process unrelated to treatment. Clinical signs of soft faeces were observed in 2/21 control, 2/18 low dose 2/18 mid dose 1/21 high dose evaluated does. The degrees varied from slightly to moderately soft faeces for up to 11 days and were ascribed to administration of 0.5 mL/kg vehicle under the conditions of this study.

The differences in the mean body weights or body weight gain values of the pregnant does evaluated at all dose levels were not toxicologically or statistically significant. The differences in the mean body weights or body weight gain values of the pregnant does evaluated at all dose levels were not toxicologically or statistically significant. Minor variations were noted, such as body weight loss from GD18 to GD27 in one mid dose female (ID3517) or lower body weight gain in one high dose female (ID4511), which were associated with decreased food consumption.
The observed differences were considered incidental and ascribed to biological variability and not to test item administration. When adjusted for the gravid uterine weight, the mean corrected body weights were similar in the control and test item treated groups at up to and including 30 mg/kg bw/day.

There were no adverse effects considered related to test item administration in the food consumption of the pregnant rabbits at up to and including 30 mg/kg bw/day. The observed variations in the food consumption of the treated animals were generally correlated with the individual body weight gain values.

Twenty five (24) New Zealand White female rabbits per dose group were inseminated and there were 21, 18, 18, and 21 litters evaluated in the control, low, mid, and high dose groups, respectively. GD0 was considered the day of insemination, and each assumed pregnant, surviving female was treated from GD6 up to GD27, followed by caesarean section and euthanasia, with necropsy, macroscopic examination and uterus weight measurement on GD28, see table 1.

The maternal reproductive parameters, number of corpora lutea, number of implantations, pre-implantation loss, early and late embryonic loss were comparable in the Control and treated groups. However, it should be noted that the early embryonic loss values appeared to be higher than Control, without attaining statistical significance, at 10 and 30 mg/kg bw/day dose levels, but the levels were well within the historic control range. There was considered to be no relationship with treatment.

The number of dead foetuses and the post-implantation loss were statistically higher in the high dose group than control (p < 0.01). The historical control range for dead foetuses has a maximum of 3.42% (Appendix 1.10) which represents typically 1 or 2 foetuses in 0-2 litters per group. In this study the high dose group incidence is 11.54%, with 1-4 dead foetuses in 12 litters, this is outside the historical range. The maximum post-implantation loss is 8.48% in the historical controls (with a maximum of 7 litters affected) and 16.24% in the high dose group (with 16 litters affected). These findings indicate a foetotoxicity effect at the high dose only, which may be related to sub-clinical maternal effects.
Subsequently the total intrauterine mortality was also higher than control, but the mean value remained comparable with the historical control range, since the early and late embryonic losses were not affected by treatment, see table 2.

Placentas were normal in all animals examined during the caesarean section. There were no test item related changes in the gravid uterine weight recorded in the treated pregnant does when compared to the controls.

There were no test item related macroscopic changes in the surviving animals (termination on GD28) administered with Bis-(2-chloroethoxy)methane through oral gavage at dose levels 3, 10, and 30 mg/kg bw/day.
Red multifocal discoloured fundic mucosa of the stomach in 1/24 Control and 1/22 High Dose females, yellow discoloration and adhesion of the firmed right lobes in the lungs, green-brown liquid material in the digestive content of the caecum in 1/23 Mid Dose, yellow discoloration of the right pulmonary lobes and yellow-red liquid (approx. 15 ml) within the thoracic cavity in 1/22 High Dose female, were macroscopically observed. Based on the incidence and lack of significant clinical adverse effects noted during the study the changes in the lungs, stomach, caecum or thoracic cavity in these surviving animals, these observations were not ascribed to test item administration in the conditions of this study.

Conclusions for Maternal Toxicity
In this study the high dose level (30 mg/kg bw/day) females did not demonstrate visible signs of maternal toxicity. However, in the preliminary study it was found that a dose level of 60 mg/kg bw/day (twice the current high dose) caused 100% mortality; so it is evident that the high dose in this current study, although there were no measured signs of maternal toxicity, it is likely that there were some sub-clinical effects of the test item on the pregnant does.





Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: related to treatment

Details on embryotoxic / teratogenic effects:
Compared to the control, lower total and litter mean number of viable foetuses was noted in the high dose group, attaining statistically significance (p < 0.01) in the total numbers of foetuses. However this difference was not considered to be test item related because the mean values remained well within the historical control ranges with a mean of 6 per litter (historical control viable foetuses mean: 7.14 with minimum: 2 and maximum: 14). There were no test item related effects on sex ratios.

There was no toxicologically significant difference in the litter means of the foetal body weight and crown-rump length at any dose level. Slightly higher than control mean foetal weight was noted in mid dose group and slightly higher mean crown-rump length was observed in low and mid dose group (p<0.05). The limit for growth retarded foetuses was calculated from the average body weight and crown rump length of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control foetuses. Based on these criteria, the number and percentage of foetuses retarded in the body weight and/or crown-rump length were evaluated. No statistically or toxicologically significant differences were observed in the treated groups compared to control, all values were considered to be normal.

There were no foetal external abnormalities related to Bis-(2-chloroethoxy)methane administration at all dose levels. Malformation was noted in of 1/139 foetus in the control, doe 1505, and 1/111 foetus in the mid dose, doe 3516, which had cleft palate. This type of malformation is represented a normal background incidence and was unrelated to the administration of the test item.

At visceral examination of the foetuses, an increased incidence of abnormalities was noted at 10 mg/kg bw/day and 30 mg/kg bw compared to control mean, the difference of litter means percentage attained statistical significance (p<0.01).

Thymic cord and small renal papilla are commonly observed variation in New Zealand White rabbits, especially associated with retarded foetal development, common in cases of maternal toxicity. These two findings were significantly elevated with a clear dose response in the mid and high dose groups. Although there were no clinical signs of maternal toxicity, these differences are classical signs of a retarded foetal development and are probably an indirect effect resulting from sub-clinical maternal toxicity.

Branched renal vein was found to have a statistically significantly higher incidence in the mid and high dose groups (p<0.05 and p<0.01 respectively). However, the mid dose group incidence was in the middle of the historical control range and the high dose group incidence was considered to not be abnormally high relative to historical control incidences. This observation is a minor variation and in isolation the observed incidence is not be considered to represent an adverse effect of the test item.

Visceral malformations were recorded such as cleft palate for one foetus in the control (1505/4) and dilated lateral brain ventricle for one foetus (4515/4) in the high dose group. One multiple malformed foetus (4505/4) was found during the visceral examination with misshapen kidney, malpositioned left adrenal gland, malpositioned left ureter, malpositioned left ovary and overriding urinary bladder in the high dose group. These malformations occurred with a low incidence in the experimental groups without any dose dependency and were considered incidental, ascribed to individual variability and not related to treatment.

In addition, minor variations including short brachiocephalic trunk and small nasal conchae were noted. Based on the low incidence and in the absence of any dose response, these findings were considered incidental ascribed to individual variability and not related to treatment.

At skeletal examination of the foetuses, an increased incidence of abnormalities was noted at 10 mg/kg bw/day and 30 mg/kg bw compared to control mean, the difference of litter means percentage attained statistical significance.

The incidence of dumbbell shaped vertebrae and/or dumbbell, bipartite or incomplete vertebrae ossification, unossified pubis and unossified talus were significantly higher (p<0.01) in the high dose group. The differences were considered to be related to treatment and are related to minimally retarded or incomplete ossification. The higher incidence of skeletal variations in the mid and high dose groups were related to finding which are commonly associated with maternal toxicity, with retarded foetal development.

Fused sternal bodies were recorded for one foetus in the mid dose group and for three foetuses in the high dose group (2 in one litter and one in another), the differences did not attain statistically significance. The historical data for this observation shows that it is common to have one foetus in one or two groups per study. When the occurrence of fused sterna is a significant malformation, it is associated with rib malformations; in this study there were no associated rib malformations. Taking into account the low incidence and the lack of associated skeletal changes, the observation was ascribed as biological variability and not a malformation related to treatment.

Variations such as 4 or less sternal body, 13 or less coccygeal body, xiphoid cartilage hole or bipartite, misaligned or asymmetric sternal bodies, or malformations including malformed vertebrae, fused transverse processes, or cleft palate were observed. In the absence of a dose response or of statistically significance differences between the treated and control groups, these variations were ascribed to biological variability and not considered correlated with Bis-(2-chloroethoxy)methane administration under the conditions of this study.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Bis-(2-chloroethoxy)methane administered at 0.5 mL/kg in PEG400 daily from gestation day 6 to 27 to artificially inseminated New Zealand White rabbits by oral gavage at dose levels of 3, 10 and 30 mg/kg bw/day did not lead to any clinical evidence of maternal toxicity. Therefore NOAELmaternal toxicity is 30 mg/kg bw/day. However, the high dose of 30 mg/kg bw/day is only half of the dose of 60 mg/kg bw/day which caused 100% mortality in the preliminary study, so sub-clinical maternal toxicity in the current high dose group is probable.
At reproduction toxicity evaluation no test item related effects were observed on the maternal reproductive parameters, including numbers of corpora lutea, implantation sites or embryonic loss (early or late). Therefore the NOAELembryotoxicity is 30 mg/kg bw/day. Higher numbers of dead foetuses and hence post-implantation loss were observed in the high dose group. Therefore NOAELfoetotoxicity is 10 mg/kg bw/day. As mentioned above the effects observed at 30 mg/kg bw/day may be related to sub-clinical maternal toxicity.
At external foetal examination there were no effects of treatment observed.

At visceral foetal examination there were increases in the incidence of variations at 10 and 30 mg/kg bw/day. Small renal papilla and thymic cord were at a relatively high incidence, particularly in the high dose, these are changes commonly related to retarded foetal development. There was no significant increase in visceral malformations.
At skeletal examination there were increases in the incidence of variations at 10 and 30 mg/kg bw/day. At 30 mg/kg bw/day dumbbell shaped vertebrae and/or dumbbell, bipartite or incomplete vertebrae ossification, unossified pubis and unossified talus were observed. The variations with increased incidences are changes commonly related to retarded foetal development. There was no significant increase in skeletal malformations.
At a dose level close to a lethal maternal dose there was no overt maternal toxicity as measured by the usual maternal parameters measured in this type of study. There were visceral and skeletal variations characteristic of retarded foetal development at 10 and 30 mg/kg/day and some foetotoxicity at 30 mg/kg bw/day; the observed changes are commonly associated with maternal toxicity. There were no malformations ascribed to treatment with the test item. Therefore NOAEL teratogenecity is 30 mg/kg bw/day.

In summary this study, from the observations made in the does and their foetuses, the following no-observed-adverse-effect levels were derived:

NOAELmaternal toxicity: 30 mg/kg bw/day
NOAELembryotoxicity: 30 mg/kg bw/day
NOAELfoetotoxicity: 10 mg/kg bw/day
NOAELteratogenecity: 30 mg/kg bw/day
Executive summary:

The objective of this study was to assess the effects of the test item on pregnant females and on the developing conceptuses and provide general information concerning the effects of prenatal exposure on the pregnant test animals and on the developing organism, to include assessment of maternal effects as well as death, structural abnormalities, or altered growth in the foetus. The study was performed in the rabbit, which is the preferred non-rodent species. Dosing formulations were administered daily by oral (gavage) to artificially inseminated, assumed pregnant New Zealand White rabbits from GD6 to GD27, where the day of artificial insemination was regarded as Gestation Day 0 (GD0), according to the study design below:

Group no./ Designation

Dose Level

(mg/kg bw/day)

Concentration

 (mg/mL)

Dose volume

(mL/kg)

Number of inseminated females

Does ID

1/Control

0

0

 

 

0.5

24

1501-1524

2/Low dose

3

6

24

2501-2524

3/Mid dose

10

20

24

3501-3524

4/High dose

30

60

24

4501-4524

The control group was treated with the vehicle only (Polyethylene glycol 400). The dose levels were set by the Sponsor in consultation with the Study Director based on available data, including CiToxLAB study code 12/094-028N and 12/094-105NE, with the aim of determining the MTD and selecting dose levels for the main study. These chosen dose levels should induce toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose. In the preliminary study to determine the MTD, 100% mortality was observed at doses of 90 mg/kg bw/day and above. The MTD in non-pregnant female New Zealand White rabbits, treated daily for at least 21 days, was considered to be slightly below 62.5 mg/kg bw/day. In the range finding study with pregnant female New Zealand White rabbits, the high dose of 60 mg/kg bw/day was considered to be above the MTD causing 100% mortality. At 30 mg/kg bw/day some toxicity was observed and no effects were seen at 15 mg/kg bw/day. The dose levels selected for a main developmental toxicity study in New Zealand white pregnant rabbits in agreement with the Sponsor were 3, 10 and 30 mg/kg bw/day

The clinical symptoms, mortality, necropsy findings, body weight and body weight gain, food consumption and reproduction data of the does were evaluated. Shortly before caesarean section, the females were euthanized, the uterine content was examined, and the foetuses were evaluated for soft tissue and skeletal changes. The sex, body weight and crown-rump length of the foetuses were assessed; the placentas were examined externally and the external, visceral and skeletal abnormalities of foetuses were recorded and evaluated.

Formulations were prepared fresh prior to administration to animals or up to four days prior in the vehicle; appropriate samples were analysed using a validated gas-chromatographic method. Stability, homogeneity and measured concentrations were all within the acceptance criteria.

No clinical signs or mortality considered related to Bis-(2-chloroethoxy)methane were noted during the study following administration to artificially-inseminated females at dose levels up to and including 30 mg/kg bw/day. There were deaths unrelated to treatment, but all groups had 18 - 21 evaluable litters, therefore the study is considered to be valid. It is noted that in the preliminary study a dose level of 60 mg/kg/day caused 100% maternal lethality, so sub-clinical maternal toxicity at 30 mg/kg/day was probable.

No statistically or toxicologically significant changes were observed during the study in the maternal body weight, body weight gain, or food consumption. There were no test item related effects on any of the maternal reproductive parameters examined during the study. The numbers of corpora lutea and implantation sites were similar in the treated groups compared to control animals.

The number of dead foetuses and the post-implantation losses were statistically higher in the high dose group than control (p < 0.01). The number of affected litters, and the percentage losses were outside the historical control range. These findings indicate a foetotoxicity effect at the high dose only, which may be related to maternal effects.

Placentas were normal in all animals examined during the caesarean section. There were no test item related changes in the gravid uterine weight recorded in the treated pregnant does when compared to the controls.

There were no test item related macroscopic changes at necropsy in the surviving does (termination on GD28).

There were no test item related effects on viable foetuses per litter or on sex ratios.

There was no toxicologically significant difference in the litter means of the foetal body weight and crown-rump length at any dose level.

There were no foetal external abnormalities related to Bis-(2-chloroethoxy)methane administration at any dose level.

At visceral examination of the foetuses, an increased incidence of abnormalities was noted at 10 mg/kg bw/day and 30 mg/kg bw/day compared to control mean, the difference of litter means percentage attained statistical significance (p<0.01).

Thymic cord and small renal papilla are commonly observed variations in New Zealand White rabbits, especially associated with retarded foetal development, common in cases of maternal toxicity. These two findings were significantly elevated with a clear dose response in the mid and high dose groups, with a high incidence in the high dose group. Although there were no clinical signs of maternal toxicity, these differences are classical signs of a retarded foetal development and are probably indirect effects resulting from sub-clinical maternal toxicity.

Branched renal vein was found to have a statistically significantly higher incidence in the mid and high dose groups (p<0.05 and p<0.01 respectively). However, the mid dose group incidence was in the middle of the historical control range and the high dose group incidence was considered to not be abnormally high relative to historical control incidences. This observation is a minor variation and in isolation the observed incidence is not be considered to represent an adverse effect of the test item.

The visceral abnormalities with increased incidences were all classed as variations; there were no increases in malformations. The increased variations were mainly changes that are usually associated with retarded development.

At skeletal examination of the foetuses, an increased incidence of abnormalities was noted at 10 mg/kg bw/day and 30 mg/kg bw/day compared to control mean, the difference of litter means percentage attained statistical significance (p<0.05 or p<0.01).

The incidence of dumbbell shaped vertebrae and/or dumbbell, bipartite or incomplete vertebrae ossification, unossified pubis and unossified talus were significantly higher (p<0.01) in the high dose group. The differences were considered to be related to treatment and are related to minimally retarded or incomplete ossification.

The skeletal abnormalities with increased incidences were all classed as variations; there were no increases in malformations. The increased variations were mainly changes that are usually associated with retarded development. The higher incidence of skeletal variations in the mid and high dose groups were related to finding which are commonly associated with maternal toxicity, with retarded foetal development.

There were no statistically significant increases in skeletal malformations. Numerical differences in low incidences were not considered to be malformations related to treatment.

Conclusion

Bis-(2-chloroethoxy)methane administered at 0.5 mL/kg in PEG400 daily from gestation day 6 to 27 to artificially inseminated New Zealand White rabbits by oral gavage at dose levels of 3, 10 and 30 mg/kg bw/day did not lead to any clinical evidence of maternal toxicity. Therefore NOAELmaternal toxicity is 30 mg/kg bw/day. However, the high dose of 30 mg/kg bw/day is only half of the dose of 60 mg/kg bw/day which caused 100% mortality in the preliminary study, so sub-clinical maternal toxicity in the current high dose group is probable.

At reproduction toxicity evaluation no test item related effects were observed on the maternal reproductive parameters, including numbers of corpora lutea, implantation sites or embryonic loss (early or late). Therefore the NOAELembryotoxicity is 30 mg/kg bw/day. Higher numbers of dead foetuses and hence post-implantation loss were observed in the high dose group. Therefore NOAELfoetotoxicity is 10 mg/kg bw/day. As mentioned above the effects observed at 30 mg/kg bw/day may be related to sub-clinical maternal toxicity.

At external foetal examination there were no effects of treatment observed.

At visceral foetal examination there were increases in the incidence of variations at 10 and 30 mg/kg bw/day. Small renal papilla and thymic cord were at a relatively high incidence, particularly in the high dose, these are changes commonly related to retarded foetal development. There was no significant increase in visceral malformations.

At skeletal examination there were increases in the incidence of variations at 10 and 30 mg/kg bw/day. At 30 mg/kg bw/day dumbbell shaped vertebrae and/or dumbbell, bipartite or incomplete vertebrae ossification, unossified pubis and unossified talus were observed. The variations with increased incidences are changes commonly related to retarded foetal development. There was no significant increase in skeletal malformations.

At a dose level close to a lethal maternal dose there was no overt maternal toxicity as measured by the usual maternal parameters measured in this type of study. There were visceral and skeletal variations characteristic of retarded foetal development at 10 and 30 mg/kg/day and some foetotoxicity at 30 mg/kg bw/day; the observed changes are commonly associated with maternal toxicity. There were no malformations ascribed to treatment with the test item. Therefore NOAEL teratogenecity is 30 mg/kg bw/day.

In summary this study, from the observations made in the does and their foetuses, the following no-observed-adverse-effect levels were derived:

NOAELmaternal toxicity: 30 mg/kg bw/day

NOAELembryotoxicity: 30 mg/kg bw/day

NOAELfetotoxicity: 10 mg/kg bw/day

NOAELteratogenecity: 30 mg/kg bw/day

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