Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 January 2015 to 22 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 414 compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate n°2015/5 dated 5 March 2015
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Anisole
EC Number:
202-876-1
EC Name:
Anisole
Cas Number:
100-66-3
Molecular formula:
C7H8O
IUPAC Name:
anisole
Test material form:
other: liquid stored at room temperature
Details on test material:
- Name of test material (as cited in study report): Anisole
- Chemical name: Methoxybenzene

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
- Number: 96 time-mated female rats were received at CiToxLAB France between 19 and 27 March 2015.
- Strain and sanitary status: Sprague-Dawley, Rj Han: SD (Rats CD). Upon their arrival at CiToxLAB France, the animals were given a clinical examination to ensure that they were in good condition.
- Breeder: Janvier, le Genest-Saint-Isle, France.
- Age/Weight: at the beginning of the treatment period, the females were 10-11 weeks old and had a mean body weight of 299 g (range: 229 g to 355 g). The females were sexually mature and primigravid.
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm², 48 cm x 26.5 cm x 21 cm) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen since it is preferable for pregnant animals.
Each cage contained an object for the environmental enrichment (Nylabone and cocoon) of the animals.
The cages were placed in numerical order on the racks.
- Food and water: All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 4482794 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation: the animals were acclimated to the study conditions for a period of at least 4 days before the beginning of the treatment period (arrival of the females on Day 1 or 2 p.c.).
- Allocation to group: during the acclimation period, the animals were allocated to the groups, according to a computerized stratification procedure, based on body weight recorded on Day 2 p.c., so that the average body weight of each group was similar.
- Identification: each animal was individually identified by an ear tattoo or tail tattoo (unique CiToxLAB France identity number).

ENVIRONMENTAL CONDITIONS
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
- temperature: 22 ± 2°C,
- relative humidity: 50 ± 20%,
- light/dark cycle: 12h/12h,
- ventilation: about 8 to 15 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
DOSE FORMULATION PREPARATION
The test item was administered as a solution in the vehicle.
The required quantities were mixed progressively with the vehicle in order to obtain the desired concentration.
After addition of the vehicle, the dose formulations were kept under magnetic stirring for at least 30 minutes to ensure effective solubilization of the test item.
The test item dose formulations were prepared at a frequency depending of the result of the stability study (CiToxLAB France/Study No. 42377 AHS), stored and delivered at room temperature.


VEHICLE
The vehicle was corn oil, batch No. MKBQ9948V.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations have been quantified by a validated analytical method.
The Gas Chromatography with FID detection (GC-FID) analytical method for the determination of Anisole in dose formulation samples was provide by the Sponsor and was validated at CiToxLAB France (CiToxLAB France/Study No. 42376 VAA) prior to dose formulation analysis.

The validation of the analytical method was conducted in CiToxLAB France/Study No. 42376 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.

Details on mating procedure:
- Mating: the females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c.).

Duration of treatment / exposure:
The dose formulations were administered daily from day 6 to day 20 p.c., inclusive.
Frequency of treatment:
Daily
Duration of test:
One month
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of a preliminary study of prenatal developmental toxicity by oral route (gavage) in rats (CiToxLAB France/Study No. 42378 RSR).

In this study, three groups of five pregnant female Sprague-Dawley rats received the test item daily, by oral administration (gavage) throughout gestation (Days 6 to 20 p.c.) at dose-levels of 200, 500 or 1000 mg/kg/day. An additional group of five pregnant females received the vehicle control, corn oil, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

At 1000 mg/kg/day, there were:
- toxicologically significant clinical signs, from near the mid and/or end of the treatment period (piloerection, staggering gate, round back, ventral decubitus and eyes half-closed),
- body weight loss on initiation of the treatment (-5 g vs. +9 g in controls during the period of Days 6 to 9 p.c., p<0.01) followed with lower mean body weight changes during the period of Days 15 to 21 p.c. (down to +33 g vs. +45 g in controls, p<0.05),
- reduced mean food consumption on initiation of the treatment (-36% vs. controls during the period of Days 6 to 9 p.c., p<0.01) which returned to control values thereafter,
- no effects on pregnancy parameters (pre- and post-implantation losses, number of viable fetuses),
- lower percentage of male fetuses (20.0% vs. 46.2% in controls, p<0.05) associated with a lower mean fetal body weight both in male and female fetuses (-30% vs. controls, p<0.001),
- there were no findings at external (including oral cavity) examination of the fetuses.

At 500 mg/kg/day, there were:
- a few clinical signs (ptyalism and/or reddish vaginal discharge) which were not considered to represent maternal toxicity in the absence of any effect on body weight, food consumption and pre- and post implantation losses,
- lower percentage of male fetuses (35.0% vs. 46.2% in controls, not statistically significant) associated with a lower mean fetal body weight both in male and female fetuses (-14.5% vs. controls, p<0.001),
- there were no findings at external (including oral cavity) examination of the fetuses.

At 200 mg/kg/day, there were no findings (with the exception of one female with ptyalism on Day 14 p.c.).

Therefore, 800 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing approximately a 4-fold interval (i.e. 50 and 200 mg/kg/day).


Examinations

Maternal examinations:
MORBIDITY AND MORTALITY:
Each animal was checked for mortality and morbidity once a day before and after the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS:
From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
The body weight of each female was recorded on days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

FOOD CONSUMPTION:
The quantity of food consumed by each female was recorded for the following intervals:
- days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21 p.c.
- The weight of gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy.
- Macroscopic post-mortem examination of the principal thoracic and abdominal organs.
Any macroscopic lesions observed in females were sampled and kept preserved in 10% buffered formalin (or in another appropriate fixative). As remarkable macroscopic lesions were noted in test item-treated group animals, the corresponding tissues of five control animals were sampled and preserved.
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with discernible digits.

A gross evaluation of placentas was also undertaken.



Fetal examinations:
Fetal examination was conducted in all viable fetuses from all litters. The fetal findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformations or variations.

BODY WEIGHT OF FETUSES:
The body weight of each live fetus was recorded.

SEX OF FETUSES:
The sex of each live fetus was determined at the time of hysterectomy by visual assessment of anogenital distance and was confirmed by internal examination of sexual organs at detailed dissection of the soft tissues or at evisceration.

EXTERNAL EXAMINATION:
Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

SOFT TISSUE EXAMINATION:
As soon as possible after sacrifice, approximately half of the fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed with Harrison's fluid for examination of the structures of the head.

SKELETAL EXAMINATION:
The remaining live fetuses per litter were eviscerated and then fixed with ethyl alcohol.
A detailed examination of the skeleton (bones + cartilage) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs.
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher exact probability test (proportions).

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: At termination on Day 21 p.c., there were 23, 22, 24 and 23 females with live fetuses in the groups treated at 0, 50, 200 and 800 mg/kg/day, respectively. Therefore anisole have no effect on pregnancy status.

MORTALITY: There were no unscheduled deaths.

CLINICAL SIGNS: At 50 mg/kg/day, there were no toxicologically significant findings. From 200 mg/kg/day, ptyalism was considered to be test item treatment-related and of minor toxicological importance. This effect was observed in only one female at 200 mg/kg/day and in 24/24 females at the dose level of 800 mg/kg/day. At 800 mg/kg/day, four females (D28650, D28651, D28664 and D28665) had clinical signs (round back, piloerection and/or staggering gait) which were considered to be test item treatment-related and of toxicological significance.Swollen urogenital area and reddish vaginal discharge are commonly observed findings in this species and strain of rats.

BODY WEIGHT: There were no toxicologically significant effects on mean body weight. When compared with controls, there were no toxicologically significant effects on mean body weight change at 50 mg/kg/day but a tendency towards lower gain at 200 mg/kg/day (down to -13% on Days 18 21 p.c., p<0.05). At 800 mg/kg/day and when compared with controls, there was a lower mean body weight gain (-19% on Days 6-21 p.c., p<0.001) between Days 6 and 21 p.c. (as a consequence of lower mean body weight gains during the periods of Days 15 to 21 p.c.). Taking into account the amplitudes and durations of the changes, the lower mean body weight gain at 800 mg/kg/day was considered to be test item treatment-related and toxicologically significant.

FOOD CONSUMPTION: There were no toxicologically significant effects at 50 and 200 mg/kg/day. At 800 mg/kg/day and when compared with controls, mean food consumption was markedly lower on initiation of the treatment (-20% on Days 6 to 9 p.c.) but with a return towards controls values thereafter. This finding was considered to be test item treatment-related and toxicologically significant taking into account the amplitude of the change. The statistical significance (p<0.05) for the Day 18-21 p.c. interval was considered to be of non toxicological relevance.

MACROSCOPIC post-mortem EXAMINATION: There were no findings in the control group. In the 50 mg/kg/day group, pregnant female D28609 had a mass on a mammary gland. In the 200 mg/kg/day group, pregnant female D28635 had a right kidney with dilated pelvis. In the 800 mg/kg/day group, female D28650 had a dilated right ureter/kidney, and female D28665 with implant scars had both horns with dilatation and serous content.
These findings are commonly observed in this species and strain of rats.

NET BODY WEIGHT CHANGE: At 50 or 200 mg/kg/day, and when compared with controls, there were no effects on mean gravid uterus weights, mean carcass weights and net body weight. At 800 mg/kg/day, there was a lower mean gravid uterus weight (-20% vs. controls, p<0.001) resulted in a lower mean net weight change (-17% vs. controls, not statistically significant). These findings were considered to be test item treatment-related and toxicologically significant.

HYSTERECTOMY DATA: At 50 and 200 mg/kg/day and when compared with controls, there were no test item treatment-related effects.
At 800 mg/kg/day and while not statistically significant when compared with controls, there were higher mean number of scars (0.7 vs. 0.0) and lower mean number of live fetuses (12.7 vs. 13.6), mean post implantation loss was recorded at an incidence higher than the upper limit of the Historical Control Data (10.6% vs. 6.2%), and one female (D28665) had only implantation scars. Taking into account all these findings, a test item treatment-related effect was not excluded.

Effect levels (maternal animals)

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

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
FETAL BODY WEIGHT AND SEX RATIO: When compared with controls, there were dose-related and statistically significant (p<0.001) lower mean fetal body weights at 800 mg/kg/day. The effect was of similar amplitude in both sexes (-22% in males and -21% in females vs. controls) and considered to be toxicologically significant.There was no effect on mean percentage of male fetuses (as confirmed at visceral examination). This result has to be compared with the observations made during the preliminary study (CiToxLAB/Study No. 42378 RSR) where in fetuses with a low body weight, the apparent anogenital distance was close to that of females (the sex of the fetuses being not confirmed by visceral examination) and resulted in a higher proportion of fetuses identified as females because of an anogenital distances comparable to that of females (mean percentage of male fetuses down to 20% vs. 46% in controls at 1000 mg/kg/day, p<0.001).
There were 1/312 (0.3%), 1/282 (0.4%), 5/320 (1.6%) and 5/292 (1.7%) males fetuses in control, 50, 200 and 800 mg/kg/day groups, respectively, which were incorrectly sexed by appreciation of the anogenital distance.

EXTERNAL EXAMINATION: In control, 50 and 200 mg/kg/day groups, there were no variations at external examination of the fetuses. At 800 mg/kg/day, all fetuses from all litters had discolored skin and moderate subcutaneous edema which were considered to be test item treatment-related and toxicologically significant. There were no test item treatment increased litter incidences of external malformations at fetal examination. In the controls, two litters had fetuses with external malformations: D28588-06 with omphalocele and D28571-07/13/14 with short tail. In the 200 mg/kg/day group, one litter had a fetus with external malformations: D28627-09 with microstomia, exophthalmos and open eye.

SOFT TISSUE EXAMINATION: At soft tissue examination and when compared with controls, there were no test item treatment-related effects on litter or fetal incidences of variations. There were no malformations in test item-treated groups at soft tissue examination. In the controls, two litters had fetuses with soft tissue malformations: D28571-11/13 with marked dilated cerebral ventricle and D28590-04 with marked small kidney.

CARTILAGE AND SKELETAL EXAMINATION: At cartilage examination and when compared with controls, there were a few statistical significant differences but none were of toxicological relevance. At skeletal examination and when compared with controls, there were no test item treatment-related effects on litter or fetal incidences of variations or malformations. In the control group, two litters had a malformed fetus: D28571-14 with fused sternebra(e) and D28572-13 with fused cervical arches/centrum(a), absent thoracic vertebra(e), fused thoracic centrum(a) and fused sternebra(e). In the 200 mg/kg/day group, one litter had a malformed fetus: D28627-09 with absent parietal/interparietal/supraoccipital, agnathia (absent mandible), unossified palate and branched rib(s).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Basis for effect level:
other: based on lower fetal body weight and increased external fetal variations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

CLINICAL OBSERVATIONS

Table 1: Pregnancy statuses

Dose-level (mg/kg/day)

0

50

200

800

Number of females

24

24

24

24

Females with live fetuses

23

22

24

23

Females with total resorption

0

0

0

1

Non-pregnant females

1

2

0

1

Table 2: Clinical signs

Dose-level (mg/kg/day)

0

50

200

800

Round back

 

 

 

3

(from Day 8 up to 13, Days 12, 13 and 15 to 18 or Day 20p.c.)

Piloerection

 

 

 

1

(Days 12, 13 and Days 15 to 18p.c.)

Staggering gait

 

 

 

1

(Day 13p.c.)

Swollen urogenital area

 

1

(Days 8 to 21p.c.)

 

 

Ptyalism

 

 

1

(Days 16 and 17p.c.)

24

(from Day 8 up to 21p.c.)

Reddish vaginal discharge

1

(Day 14p.c.)

1

(Day 14p.c.)

1

(Days 15 and 16p.c.)

1

(Days 15, 20 and 21p.c.)

Number of affected animals

1/24

2/24

2/24

24/24

( ) in brackets: days of occurrences.

MATERNAL TERMINAL EXAMINATIONS

Table 3: Hysterectomy data

Dose-level (mg/kg/day)

0

50

200

800

HCD

Number of females with live fetuses
at termination

23

22

24

23

185

Mean number ofcorpora luteaper animal

15.6

15.2

15.2

15.4

[14.4; 16.6]

Mean number of implantations per animal

14.3

13.8

14.1

14.3

[12.9; 14.6]

Mean pre-implantation loss (%)

8.3

8.7

7.4

7.0

[6.5; 12.7]

Mean number of live fetuses per animal

13.6

12.8

13.3

12.7 (a)

[12.5; 14.0]

Dead fetuses (%)

0

0

0

0

[0.00; 0.10]

Mean number of scars

0.0

0.0

0.0

0.7 (b)

/

Mean number of early resorptions

0.6

0.9

0.6

0.8

/

Mean number of late resorptions

0.1

0.1

0.1

0.1

/

Mean post-implantation loss (%)

5.0

7.0

5.9

10.6

[1.5; 6.2]

(a): statistically significantvs. controls in terms of total number of fetus (292vs.312, p<0.01).

(b): statistically significantvs. controls in terms of total number of scars (15vs.0, p<0.001).

HCD: Historical Control Data (control data collected from seven studies covering a period ranging from March 2013 to June 2014), [min.; max.].

/: not available in HCD.

FETAL EXAMINATIONS

Table 4: Fetal body weight and percentage of male fetuses

Dose-level (mg/kg/day)

0

50

200

800

HCD

Mean fetal body weight (g)

5.54

5.59

(+1)

5.51

(-1)

4.36#

(-21)

[5.5; 5.8]

- male fetuses (g)

5.69

5.73

(+1)

5.67

(0)

4.46#

(-22)

[5.6; 6.0]

- female fetuses (g)

5.40

5.43
(+1)

5.36
(-1)

4.27#
(-21)

[5.4; 5.7]

Mean percentage of male fetuses (%)

48.5

51.3

48.7

46.3

[42.8; 52.2]

HCD: Historical Control Data (control data collected from seven studies covering a period ranging from March 2013 to June 2014), [min.; max.].

( ): in brackets, percentage differencevs.controls (%).

Statistical significance: #: p<0.001.

Applicant's summary and conclusion

Conclusions:
The test item, Anisole, was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to time-mated female Sprague-Dawley rats at dosages of 50, 200 or 800 mg/kg/day.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 200 mg/kg/day based on clinical signs, low body weight change / food consumption at 800 mg/kg/day.
- the NOAEL for effects on fetuses was considered to be 200 mg/kg/day based on lower fetal body weight and increased external fetal variations which were considered to be secondary to the maternal toxicity recorded at 800 mg/kg/day.

Anisole did not reveal a teratogenic potential.
Executive summary:

The objective of this prenatal development toxicity study was to evaluate the potential toxic effects of the test item, Anisole, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 20 post-coitum (p.c.), inclusive).

 

Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, Anisole (batch No. FOL1428201), once daily from Day 6 to Day 20 p.c., by gavage at dosages of 50, 200 or 800 mg/kg/day. An additional group of 24 time-mated females received the vehicle, corn oil, under the same experimental conditions and acted as the control group. A dose-volume of 5 mL/kg/day was used.

 

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.

 

On the basis of the results obtained in this study:

the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 200 mg/kg/day (based on clinical signs, low body weight change / food consumption at 800 mg/kg/day),

the NOAEL for embryo-fetal development (effects on fetuses) was considered to be 200 mg/kg/day (based on lower fetal body weight and increased external fetal variations which were considered to be secondary to the maternal toxicity recorded at 800 mg/kg/day).

No teratogenic effects were observed. Only effects on fetuses were noted at the dose level of 800 mg/kg/day. These effects were considered to be secondary to the maternal toxicity. Therefore, based on the results of this test, Anisole did not reveal a teratogenic potential.