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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2008 - 22 April 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study conducted according to OECD Guideline 414 with deviations: acclimation period followed for 4 or 5 days instead of 5 days; temperature and relative humidity were not recorded for one day; single instead of duplicate analyses was performed for determination of content in control group on Day 1
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
acclimation period followed for 4 or 5 days instead of 5 days; temperature and relative humidity were not recorded for one day; single instead of duplicate analyses was performed for determination of content in control group on Day 1
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): CHIMEXANE NB
- Physical state: Brownish viscous liquid
- Analytical purity: 97.3 %
- Lot/batch No.: 0129336
- Date of receipt: 27 February 2006
- Expiration date of the lot/batch: September 2008
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 267 g (mean body weight)
- Housing: Animals were housed individually in wire-mesh cages (43.0 x 21.5 x 18.0 cm).
- Diet: A04 C powdered maintenance diet (SAFE, Augy, France) distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: About 12 cycles/h of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: dietary admixture, in A04 C powder maintenance diet
Details on exposure:
DIET PREPARATION
- For each concentration, a premix with the total amount of test item was prepared in the diet using a mixer. The final mix was prepared by dispersing the premix in the remaining diet in the mixer and then mixing for at least 10 minutes.
- Storage temperature of food: Dietary admixtures were prepared on a weekly basis and were stored in closed bags at room temperature and protected from light prior to use.

STABILITY
- Satisfactory homogeneity and stability of dietary admixtures prepared at 500 or 20000 ppm was previously demonstrated during the study (Study No. 34827 AHS), over a 14-day storage period in open feeders and in closed bags at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentration of the test item was determined in samples of each control and test item dietary admixture prepared for use on the first day of treatment of the first female and on the last day of treatment of the last female.
- Acceptance criterion: Measured concentration = nominal concentration ± 20 %.

Results: Test item concentrations in the administered dietary admixtures analyzed on the first day of treatment of the first female and on the last day of treatment of the last female remained within an acceptable range of [- 17 % to + 4 %] of variation compared to the nominal values.

Details on mating procedure:
- Impregnation procedure: Purchased timed pregnant; the females were mated at the breeder's facilities (Janvier, Le Genest-Saint-Isle, France).
- Proof of pregnancy: Detection of vaginal plug referred to as Day 0 post-coitum (p.c.).
Duration of treatment / exposure:
15 days (Days 6-20 p.c.)
Frequency of treatment:
Once daily; animals were allowed the dietary admixture ad libitum
Duration of test:
22 days (Days 0-21 p.c.)
No. of animals per sex per dose:
24 mated females/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of the results of 4-week toxicity study (Study No. 31261 TSR) in which the test item was administered by dietary admixture at the target dose-levels of 150, 450 and 1000 mg/kg bw/day.
- Rationale for animal assignment: Before the beginning of the treatment period, the animals were allocated to the groups, according to a stratified procedure based on body weight recorded on Day 2 p.c., to ensure comparatively similar mean body weights among groups.

Examinations

Maternal examinations:
CAGE SIDE & CLINICAL OBSERVATIONS: Yes
Time schedule:
- Mortality or signs of morbidity: Once a day before the start of treatment and then twice a day during treatment period, including weekends and public holidays.
- Clinical signs: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Achieved intake of the test item was calculated for the following intervals 6-9, 9-12, 12-15, 15-18 and 18-21 p.c. for each test item-treated group as follows:
D = C x FC/BW; where, D = achieved dosage (mg/kg bw/day), C = nominal concentration of active test item (ppm: mg test item/10^6 mg food), FC = mean food consumption (g/animal/day), BW = mean body weight (g)

POST-MORTEM EXAMINATIONS: Yes
- Time schedule: On Day 21 p.c., females were sacrificed by inhalation of carbon dioxide followed by cervical dislocation and were subjected to a macroscopic post-mortem examination (principal thoracic and abominal organs).

OTHER:
Preservation of tissues:
- Any macroscopic lesions observed were sampled and kept preserved in 10 % buffered formalin.
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 and distribution of implantations: Yes
- Number and distribution of early and late resorptions: Yes
- Number and distribution of uterine scars: Yes
- Number and distribution of dead and live fetuses: Yes
- Gross evaluation of placentas: Yes
Fetal examinations:
EXAMINATION OF FETUSES:
The first 20 litters/group were examined as follow:
- Live fetuses were sacrificed by a subcutaneous injection of thiopental sodium.
- Body weight of fetuses: Body weight of each live fetus was recorded.
- Sex of fetuses: Sex of each fetus [excluding the autolyzed fetus] was determined at the time of hysterectomy by visual assessment of the anogenital distance and was confirmed by examination of the sexual organs at the time of detailed dissection of the soft tissues or at evisceration.
- Dead fetuses: Dead fetus was fixed in Harison’s fluid for a possible further examination.
- External examinations: Yes [all per litter excluding the autolyzed fetus]; each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.
- Soft tissue examinations: Yes, approximately half of the live fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed in Harison’s fluid for examination of the structures of the head.
- Skeletal examinations: Yes [all the remaining litters]; eviscerated and fixed with ethyl alcohol, stained with alizarin red S and alcian blue and examined for skeletal abnormalities including the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs.

For the remaining litters, fetuses were fixed with ethyl alcohol, stained with alizarin red S and alcian blue for further possible examination.

Statistics:
- Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
- Percentage values were compared by the Fisher exact probability test.
Indices:
- Pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] X 100
- Post-implantation loss: [(Number of implantation sites - Number of live fetuses) / Number of implantation sites] X 100
- Fetal or litter incidence: (Total number of fetuses or litters with a particular finding / Total number of fetuses or litters examined)X 100
- Mean proportion of affected fetuses: (Sum of proportion of fetuses affected in each litter / Total number of litters examined) X 100
Historical control data:
Historical background data was used to compare the incidences of developmental toxicity.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnancy status:
- All mated animals were pregnant, except 2/24 control females and 1/24 test-item treated females per group, and had live fetuses at term.

Mortality and clinical signs:
- No mortality was observed.
- No clinical signs were observed except cutaneous lesions on the head in one female and misshapen ear in another female were observed at 150 mg/kg bw/day, however these two events were considered fortuitous in origin and unrelated to treatment with the test item.

Body weight and body weight change:
- Lower body weight gain was observed during the first 3 days of treatment in females receiving 1000 mg/kg bw/day when compared to controls (13 g vs. 23 g) and this effect was transient and coincided with the lower food consumption. However, the terminal body weight recorded on Gestation Day (GD) 21 was not statistically significantly different from the control value and the net body weight and the carcass weights did not differ from that of the control group. It could be concluded that the test item affected the body weight gain transiently.
- At 150 and 450 mg/kg bw/day, the body weight gain and the body weight were unaffected by the treatment with the test item.

Food consumption:
- Mean food consumption of females treated at 1000 mg/kg bw/day was lower than controls [21 g vs. 26 g from GD 6 to GD 9 (p<0.001), 24 g vs. 26 g from GD 9 to GD 12 (p<0.05) and 26 g vs. 29 g from GD 12 to GD 15 (p<0.05)] and they were considered to be test item treatment-related.
- At 150 and 450 mg/kg bw/day, the food consumption was considered to have been unaffected by the test item treatment.

Maternal necropsy findings:
- There were no necropsy findings which were attributable to the test item treatment. The single observation recorded concerned one control female with dilated and presence of serous content in the uterus; this animal was not pregnant.

Gravid uterus weight:
- Mean gravid uterus weight of test item-treated females was similar to that of the control group.

Litter data:
- Number of resorptions was similar between the test item-treated groups and the controls.
- No dead fetuses were noticed except one dead fetus at 450 mg/kg bw/day.
- No differences in the mean number of implantation sites and the number of live fetuses between the test item-treated and control groups.
- Sex ratio and mean fetal body weight were similar in the control and the treated groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
External observations:
- Total of 236, 249, 257 and 231 fetuses from the control, 150, 450 and 1000 mg/kg bw/day groups were examined. Apart from one fetus of the control group with omphalocele and a dead fetus at 450 mg/kg bw/day which was autolyzed, there were no remarkable external findings in any of the fetuses examined.

Soft tissue observations:
- Total of 112, 118, 123 and 110 fetuses from the control, 150, 450 and 1000 mg/kg bw/day groups were examined.
- Fetal soft tissue variations and malformations observed were randomly distributed within the groups including the control. Furthermore, as the incidence of the observations was very low and/or incidences of variations were within historical background data (dilated renal pelvis or dilated ureter, short or absent innominate artery), they were considered to be unrelated to treatment.

Skeletal and cartilage observations:
- Total of 124, 131, 134 and 121 fetuses from the control, 150, 450 and 1000 mg/kg bw/day groups were examined.
- As the incidences of skeletal and cartilage variations and malformations were low, recorded with similar frequency among the control and treated groups and in the absence of statistical differences, they were considered to be unrelated to treatment.

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 7.8.2/1: Mean achieved dosages (mg/kg bw/day) between gestation Days 6 and 21


 






























Concentration (ppm)



1878



5634



12519



GD 6 to 21



159



464



992



Target dose-levels (mg/kg bw/day)



150



450



1000



(% from target dose-level)



+ 6 %



+ 3 %



- 1 %



 


GD: gestation day


 


Mean achieved dosages increased in a nearly dose-proportional manner and mean values were very close to the targeted dose-levels of 150, 450 and 1000 mg/kg bw/day.


 


Table 7.8.2/2: Body weight and body weight change (g)


 























































Dose-level (mg/kg bw/day)



0



150



450



1000



BW, GD 6



269



267



265



264



BW, GD 21



418



421



414



404



BWG, GD 6-9



23



20



22



13 #



BWG, GD 6-21



+149



+154



+149



+139



Carcass weight



324.8



323.4



314.3



312.3



Net weight change from GD 6



+55.9



+56.6



+49.4



+48.0



 


GD: gestation day, BW: body weight, BWG: body weight gain, #: p<0.001.


 


Table 7.8.2/3: Food consumption


 








































































































Dose-level (mg/kg bw/day)



 



0



150



450



1000



Days 2-6



Mean



23 d



22



23



22



SD



2



3



3



2



Days 6-9



Mean



26 d



25



24



21 #



SD



3



3



3



4



Days 9-12



Mean



26 d



27



27



24*



SD



4



3



3


 



4



Days 12-15



Mean



29 d



29



28



26*



SD



3



3



2



3



Days 15-18



Mean



30 d



30



30



28



SD



3



4



2



4



Days 18-21



Mean



28 d



28



26



27



SD



3



5



5



3



 


No. of animals: 22 for control and 23 for test item treated groups, mean: mean values/grams per day, d=ANOVA + Dunnett-test, * = p<0.05, # = p<0.001

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE NB was considered to be 5634 ppm (equivalent to 450 mg/kg bw/day) for maternal toxicity and the No Observed Effect Level (NOEL) of CHIMEXANE NB was considered to be 12519 ppm (equivalent to 1000 mg/kg bw/day) for developmental toxicity in Sprague-Dawley rats.
Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, CHIMEXANE NB was administered by dietary admixture to groups of mated female Sprague-Dawley, Rj Han: SD rats (24/dose) at the dose levels of 0, 1878, 5634 and 12519 ppm (equivalent to target dose-levels of 0, 150, 450 and 1000 mg/kg bw/day) from Days 6 to 20 post-coitum. Clinical signs and mortality were checked daily and maternal body weight and food consumption were recorded at designated intervals. On Day 21 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, numbers of implantations, uterine scars, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations.


 


All mated animals were pregnant except 2/24 control females and 1/24 test item-treated females per group. No deaths and no clinical signs related to treatment with the test item were recorded. The body weight gain and food consumption of animals at 1000 mg/kg bw/day were transiently lower than controls. However, the terminal body weight was not statistically significantly different from controls. None of the litter parameters evaluated were affected and there were no fetal external findings with treatment-relationship in any of the fetuses examined. No findings at visceral or skeletal (including cartilage) fetal examinations were considered to be related to the test item at the concentrations tested.


 


Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE NB was considered to be 5634 ppm (equivalent to 450 mg/kg bw/day) for maternal toxicity and the No Observed Effect Level (NOEL) of CHIMEXANE NB was considered to be 12519 ppm (equivalent to 1000 mg/kg bw/day) for developmental toxicity in Sprague-Dawley rats.