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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:
2016-05-04 to 2016-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE, 1729 MCA0
- Expiration date of the lot/batch: 07 December 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (between +15 and +25 °C)
- Stability of the test substance in the solvent/vehicle: 96 hours at concentrations of 0.2 to 50 mg/mL

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, 01400 Chatillon sur Chalaronne, France
- Age at study initiation: 17 to 19 weeks old
- Weight at study initiation: 3 to 4.5 kg
- Housing: Females were individually housed in composite plastic and metal cages in compliance with European Regulations (Directive 2010/63/EU)
- Diet: ad libitum, Pelleted complete rabbit diet (3409 CRL, KLIBA) sterilised by irradiation and analysed for chemical and bacterial contaminants.
- Water: ad libitum, Softened and filtered (0.2 µm) mains drinking water ad libitum (via an automatic watering system or via bottles).
- Acclimation period: 6 days between animal arrival and the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): > 35 %
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared as a solution in the vehicle at concentrations of 2, 6 and 18 mg/mL according to Standard Operating Procedures of the Test Facility. It was prepared at least twice weekly. For each concentration, the test item and then the vehicle were weighed and then stirred magnetically in a container until the test item was completely dissolved.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples were taken (2 x 2 mL) according to the above table from each formulation, on a single occasion during the treatment of the main study phase only (except for the group 2 formulation which was taken on additional occasions for accuracy (middle) only due to an initial achieved concentration not in agreement with the theoretical values. The samples were stored at room temperature (between +15 and +25 °C). One set of samples (1 x 2 mL) was analysed at the Test Facility using a validated method.
The duplicate formulation sample (1 x 2 mL) for the nominal 2 mg/mL concentration taken from the first preparation was analysed due to an out of specification value for the first aliquot. Remaining samples were discarded at the end of the stability period.
All analyzed samples for formulations prepared at nominal concentrations of 2, 6 and 18 mg/mL of 2,2’,2”-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol in vehicle water for injection, taken from each preparation on the first day of treatment, including the vehicle, were in agreement with acceptance criteria (± 10 %) except formulation of the low dose group at 2 mg/mL. The deviations from the nominal concentrations of Mid and High dose group ranged between -4.2 % and 7.8 % and were thus within acceptance criteria.
No test item was detected in the vehicle sample.
The first formulation for group 2 (Low dose) was outside acceptance criteria as confirmed by duplicate samples analysis. New preparation was made on first day of treatment which was outside acceptance criteria with a deviation of -13.6 %. On fourth day of treatment the preparation method was improved by addition of vehicle by portion in a beaker and stirred manually between each addition. This last preparation of the formulation was slight below the acceptance criteria -10.5 % which was considered acceptable.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
From day 6 to day 28 of gestation
Frequency of treatment:
once daily
Duration of test:
Until termination on day 29
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of the DRF phase.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: ovaries and uterus; abnormal organs detected via macroscopic examination
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 live foetuses per litter
- Skeletal examinations: Yes: all live foetuses per litter
- Head examinations: Yes: half per litter
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon
- the normality of the data distribution tested by the Shapiro-Wilk's test
- the homogeneity of the variances across groups tested by the Bartlett's test.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there is a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means are not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis is significant.
Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 is significant.
Microsoft Excel ® (2003 or higher) was employed to present certain results.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Consistent with an effect on food consumption, there was a test-item related higher frequency of reduced/no faecal output in 180 mg/kg/day treated group compared with other groups from G 6 to G 29.
There was no other treatment related clinical signs in any group.
Incidental clinical signs such as sore, scab, pale faeces and red vaginal discharge were observed in the groups given the vehicle, 60 and/or 180 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no treatment-related mortality in any group.
Female no. 111 from the control group was sacrificed on G 16 for ethical reason after trauma that occurred during the dosing procedure.
One female given 20 mg/kg/day (no. 135) was found dead on G 22 after treatment. Just after treatment, red traces were noted on the dosing tube and the female had laboured breathing. At necropsy, all right lobes of the lungs were uncollapsed and had many dark foci. Transparent fluid was also present in the thoracic cavity. Therefore the cause of death was considered to be associated with the administration procedure (gavage error).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment-related and statistically significant lower overall mean body weight gain for females given 180 mg/kg/day between G 6 and G 29 when compared with the control. The effect was principally due to a body weight loss during the first 3 dosing days (-177.4 g between G 6 and G 9). Recovery was noted from G 9 but absolute mean body weight tended to remain slightly lower (approximately 5 %) through to termination.
There was no obvious test item-related effect on body weight gain in the other groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was an overall dose-related and statistically significant lower mean food consumption for females given 60 and 180 mg/kg/day between G 6 to G 29 (130.1 and 93.3 g/day, respectively) when compared with control group (145.3 g). The effect on food consumption at 180 mg/kg/day was more pronounced from G 6 to G 15.
There was no obvious treatment related effect on mean food consumption at 20 mg/kg/day.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Consistent with lower mean foetal weight, mean gravid uterus weight was slightly (not statistically significant) lower in the 180 mg/kg/day group compared with the control.
There was no test item-related effect on mean gravid uterus weight in the lower dose groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related maternal macroscopic changes.
There were 6, 9, 8 and 5 females given vehicle, 20, 60 and 180 mg/kg/day with one or several cysts on the oviduct(s). These findings are part of the background of changes and incidental.
Other incidental findings were observed such as abnormal shaped uterus, sore/crust and irregular surface of the liver.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The pre-implantation data (corpora lutea count, number of implantations and the corresponding percentage pre-implantation loss) were comparable in all groups. There was no test item-related effect on embryo-foetal survival in any group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 19, 21, 21 and 19 pregnant females at terminal caesarean in the control, 20, 60 and 180 mg/kg/day groups, respectively, all of which had viable foetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean foetal weight was slightly but statistically significantly lower in the 180 mg/kg/day group (38.3 g) when compared with the control (42.2) and below the historical control data range of main studies (39 g – 42.1 g). Dam No. 184, which lost 96 g from G 6 to G 29, had ten considerably light foetuses with multiple malformations.
There was no test item-related effect on mean foetal weight in the lower dose groups.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no test item-related influence on foetal sex ratio in any group.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 2 (2), 1(1) and 1(1) foetuses (litters) with external malformation in the 180, 60 and 20 mg/kg/day groups, respectively, compared with none in the control group, none of which were attributed to the test item.
In the 180 mg/kg/day group, one foetus (dam no. 168) had an omphalocele and the other (dam no. 188) had a malrotated hindlimb. The foetus in the 60 mg/kg/day group (dam no. 148) had spina bifida and the other in the 20 mg/kg/day group (dam no. 144) had anencephaly.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 1 (1), 3 (3) and 11 (2) foetuses (litters) with skeletal malformations in the control, 60 and 180 mg/kg/day groups, compared with none in the 20 mg/kg/day group, none of which were attributed to the test item.
The foetus in the control group (dam no. 106) and one from the 60 mg/kg/day group (dam no. 159 had fused sternebrae. The second foetus from the 60 mg/kg/day group (dam no. 148) had confirmation of the spina bifida noted externally in the lumbar/sacral area and the other (dam no. 152) had multiple abnormalities of the cervical and thoracic vertebrae leading to scoliosis. In the 180 mg/kg/day group, 10 of the malformed foetuses came from a single litter (dam no. 184) all of which had a syndrome of atypical incomplete ossification affecting one or more regions of the skeleton (skull, sternebrae, ribs, scapulae, forelimbs and hindlimbs bones and cervical, sacral, lumbar or caudal vertebrae). The other foetus in the 180 mg/kg/day group (dam no. 177) had multiple abnormalities of the cervical vertebrae.
There was a higher incidence of delayed ossification of the extremities in the 180 mg/kg/day group when compared with other groups such as incomplete or unossified forepaw bones (metacarpal and phalanx), hindpaw (metatarsal, phalanx and tarsal bone) and stennebrae.
The incidences of the remaining skeletal anomalies and variations were comparable with the concurrent control and/or historical control data and were considered incidental.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 3 (3) and 1(1) foetuses (litters) with visceral malformations in the 20 and 180 mg/kg/day groups, respectively, compared with none in the control and 60 mg/kg/day groups, none of which were attributed to the test item.
In the 180 mg/kg/day group, the foetus (dam no. 184) had marked dilated renal pelvis. In the 20 mg/kg/day group, one foetus (dam no. 139) had a three-chambered heart with dilated aortic arch and atretic pulmonary trunk, the second (dam no. 138) had a unilateral retinal fold and the other (dam no. 144) had gross disruption of the brain associated with the anencephaly noted externally.
Other less severe visceral anomalies occurred without dose-dependency or are part of the background of changes noted for this strain of rabbit.
Details on embryotoxic / teratogenic effects:
Foetal examinations revealed a total of 1 (1), 3 (3), 3 (3) and 13 (4) malformed foetuses (litters) in the control, 20, 60 and 180 mg/kg/day groups, respectively, none of which were attributed to the test item due to the lack of any pattern or dose-related increased incidence of any of the findings, which are for the majority also part of the historical control data. In addition, there was no other evidence of a teratogenic potential of the test item with respect to embryo-foetal viability.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Overall developmental toxicity

Key result
Developmental effects observed:
no
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

The numbers of foetuses (litters) submitted to the different examinations were as follows:

Group

1

2

3

4

External examination

178 (19)

209 (21)

200 (21)

181 (19)

Internal examination

 

 

 

 

body

178 (19)

209 (21)

200 (21)

181 (19)

head

83 (19)

97 (20)

96 (21)

86 (19)

Skeletal examination

 

 

 

 

body

178 (19)

209 (21)

200 (21)

181 (19)

head

95 (19)

112 (21)

104 (21)

95 (19)

 

Summary of malformations – Individual descriptions:

Dose level (mg/kg/day)

Female number

Foetus number(s)

Malformation(s)#

0

106

9

Sternebra 3rdto 5thfused

20

138

3

Retinal fold, right eye

139

5

Heart, Three-chambered: presence of a single ventricle and two atria, and right A-V valve appeared closed.

Dilated aortic arch with atretic pulmonary trunk and ductus arteriosus

144

4

Anencephaly with malformed brain: gross disruption with left and right hemispheres misshapen and lying above midbrain

60

148

9

Spina bifida with 4thto 6thlumbar arches and 1stto 3rdsacral arches malpositioned

152

5

Vertebra, multiple abnormalities: 1stcervical centrum bipartite, 4thand 5thcervical centra fused and misshapen, 6thand 7thcervical centra small and hemicentric; odontoïd process unossified; 1stthoracic centrum small and fused with 2ndthoracic centrum, 2ndthoracic centrum misaligned; 1stbilateral cervical arches misshapen and right incomplete ossification, 5thand 7thleft cervical arches small, 5thright cervical arch absent, 2ndleft thoracic arch small; scoliosis

159

4

Sternebra 2ndto 5thfused

180

168

10

Omphalocele

177

9

Cervical vertebra, multiple abnormalities: 2ndcentrum small, hemicentric and fused with odontoïd process; 2ndbilateral arches small

184

2

Skull, Multiple abnormalities: all bones atypical incomplete ossification and misshapen

Vertebral column, multiple abnormalities: from 3rdcervical to 14thcaudal, all sternebrae and scapulae atypical incomplete ossification

3

Multiple skeletal abnormalities: scapulae, forelimb bones, hindlimb bones, vertebra column from 2ndcervical to 14thcaudal,  ribs and sternebrae atypical incomplete ossification

4

Vertebra, multiple abnormalities: 6thand 7thlumbar and 1stsacral atypical incomplete ossification

5

Vertebra, multiple abnormalities: 3rdcervical to 14thcaudal atypical incomplete ossification

Kidney, Dilated renal pelvis (right), marked

6

Vertebral column, multiple abnormalities: from 2ndcervical to 14thcaudal and all sternebrae atypical incomplete ossification

7

Vertebral column, multiple abnormalities: from 2ndcervical to 14thcaudal and all sternebrae atypical incomplete ossification

8

Vertebra, multiple abnormalities: 2ndcervical to 3rdcaudal atypical incomplete ossification

9

Vertebra, multiple abnormalities: 3rdcervical to 14thcaudal atypical incomplete ossification

10

Skull, multiple abnormalities: zygomatic arches, frontals and parietals atypical incomplete ossification; interparietal unossified

Vertebra, multiple abnormalities: 3rdcervical to 14thcaudal atypical incomplete ossification

11

Vertebral column, multiple abnormalities: from 3rdcervical to 3rdcaudal and scapulae atypical incomplete ossification

188

14

Malrotation of right hindlimb

#: including external, visceral and skeletal examinations.

 

Applicant's summary and conclusion