Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June 2016 to 28 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification Reaction products of tall oil fatty acids with diethylenetriamine and maleic anhydride
Appearance Dark brown viscous liquid/paste
Batch Development sample 15-11-1
Purity/Composition UVCB
Test item storage At room temperature
Stable under storage conditions until 19 November 2017 (expiry date)

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain: Crl:WI(Han)
- Age at study initiation: 10-14 weeks
- Sex: Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
- Weight at study initiation: weighed on day 2: 172 to 240 g
- Fasting period before study: none
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm), Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material
- Feed: Free access to pelleted rodent diet ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
prepared daily within 6 hours prior to dosing. Formulations were heated to a maximum of 63.8°C for a maximum duration of 34 minutes to obtain visual homogeneity. Formulations were allowed to cool down below 40°C before dosing. Adjustment was made for specific gravity of the substance and vehicle. No correction was made for the purity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on lab trial and sponsor information
- Dose volume 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples were taken of all concentrations on one occasion during treatment and assessed for accuracy and homogeneity. Samples were diluted with THF in order to obtain solutions of 0.1% formic acid in 50/50 (v/v) THF/water and concentrations within the calibration range. During the development of the analytical method stock solutions were stable for 12 hours when stored at room temperature.
HPLC with MS dectection
Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
Detector: Xevo TQ-S mass spectrometer (Waters)
Column: Acquity UPLC BEH C8, 50 mm × 2.1 mm i.d., dp = 1.7 µm (Waters)
Column temperature: 40°C ± 1°C
Injection volume: 5 µL
Mobile phase: 0.1% formic acid in 90/10 (v/v) acetonitrile/water
Flow 0.6 mL/min
MS detection
Ionisation source ESI+
Cone voltage 20 V
Collision energy 30eV
Quantitation sum of m/z 628.5 --> m/z 306 and m/z 630.4 --> m/z 308

Calibration range: 5 - 100 µg/L --> quadratic (r>99%)
QC samples: 1mg/g 109% ; 200 mg/g 99-104%
Accuracy: 93-106%
Homogeneity: CV 2.7-2.8%
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
day 6-20 post-coitum
Frequency of treatment:
daily
Duration of test:
day 2-21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose-range finding study
Four groups of 6 females were exposed to 0, 100, 300 and 1000 mg/kg for Days 6 to 20 post-coitum inclusive by oral gavage. These dose levels were based on a 14-day pilot study in which no toxicity was observed with treatment up to 1000 mg/kg. In a 90-day repeated dose study no effects on clinical signs, body weight or food consumption were observed by treatment up to 1000 mg/kg in the first weeks after dosing.
In this study no effects were found related to maternal or developmental toxicity up to 1000 mg/kg bw.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (mortality/viability twice daily) form day 2 post-coitum onwards

BODY WEIGHT: Yes
- Time schedule for examinations: on day 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: No quantative examination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post-coitum
- Organs examined: external, thoracic and abdominal examination, with special attention being paid to the reproductive organs

OTHER: ovary and uterine horn of all animals
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
•The number of corpora lutea.
•The weight of the (gravid) uterus.
•The number and distribution of live and dead fetuses.
•The number and distribution of embryo-fetal deaths (early and late resorptions).
Fetal examinations:
- Weight of each fetus.
- Sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- External examinations: Yes: [all per litter, including late resorptions]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Dunnett-test , Steel-test, Fisher Exact-test, Mann Whitney test, Kruskal-Wallis nonparametric ANOVA test, Dunn’s test
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance
Indices:
Pre-implantation loss (%), Post-implantation loss (%), Viable fetuses affected/litter (%)

Historical control data:
yes included in an appendix to the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during the observation period. Alopecia was noted for single females of the control, 300 and 1000 mg/kg groups. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.

dose mg/kg bw 0 100 300 1000
DAY 2 MEAN 198 196 197 198
ST.DEV. 16.8 13.7 13.2 15.1
N 22 22 22 21
.
DAY 6 MEAN 214 211 212 214
ST.DEV. 18.1 14.4 13.7 15.8
N 22 22 22 21
.
DAY 9 MEAN 222 221 220 222
ST.DEV. 18.8 15.2 13.4 17.2
N 22 22 22 21
.
DAY 12 MEAN 237 236 235 237
ST.DEV. 19.4 16.6 13.8 18.1
N 22 22 22 21
.
DAY 15 MEAN 251 249 247 248
ST.DEV. 21.8 17.6 15.2 19.3
N 22 22 22 21
.
DAY 18 MEAN 278 278 273 275
ST.DEV. 24.4 20.4 16.9 21.3
N 22 22 22 21
.
DAY 21 MEAN 309 313 305 306
ST.DEV. 27.2 25.3 20.7 23.9
N 22 22 22 21
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
dose mg/kg bw 0 100 300 1000
DAYS 2-6 MEAN 19 19 19 19
ST.DEV. 2.3 2.6 1.8 1.9
N 22 22 22 21
DAYS 6-9 MEAN 18 17 17 18
ST.DEV. 2.6 2.7 2.8 3.2
N 22 22 22 21
DAYS 9-12 MEAN 20 21 20 20
ST.DEV. 2.1 2.0 2.0 2.3
N 22 22 22 21
DAYS 12-15 MEAN 22 21 21 21
ST.DEV. 3.1 2.2 1.9 2.3
N 22 22 22 21
DAYS 15-18 MEAN 20 20 20 20
ST.DEV. 2.6 2.2 2.3 2.3
N 22 22 22 21
DAYS 18-21 MEAN 21 21 21 21
ST.DEV. 2.0 1.8 1.8 2.1
N 22 22 22 21
MEAN OF MEANS 20 20 19 20
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not quantitative assessed. No effects reported
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:
no effects observed
Description (incidence and severity):
uterus weight: no treatment related effects (average 68.7, 73.1, 69.0 and 65.7 g at 0, 100, 300 and 1000 mg/kg bw)
Gross pathological findings:
no effects observed
Description (incidence and severity):
Alopecia observed in 1 animal in control, at 300 and 1000 mg/kg bw was considered not toxicologically relevant (confirming the clinical sign observed during the in-life phase)
dose mg/kg bw 0 100 300 1000
Animals examined 22 22 22 22
Animals Without findings 21 22 21 21
Animals affected 1 0 1 1
Skin Alopecia 1 0 1 1
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
none observed
DOSE GROUP : 1 2 3 4
-----------------------------------------------------------------------------------------------------------------------------------
NO. % NO. % NO. % NO. %
FEMALES ON STUDY 22 22 22 22
FEMALES THAT ABORTED
OR DELIVERED 0 0.0 0 0.0 0 0.0 0 0.0

Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related effects
pre-implantation loss: 34, 4, 5 and 14 at 0, 100, 300 and 1000 mg/kg bw
postimplantation loss: 13, 19, 21 and 27 at 0, 100, 300 and 1000 mg/kg bw
The statistically significantly lower pre-implantation loss at 100 and 300 mg/kg were considered to be caused by a relatively high concurrent control value (11.2% compared to historical control mean value of 6.2%)

IMPLANTATION SITES 0 100 300 1000
MEAN 10.6 11.4 11.2 10.6
S.D. 2.57 1.14 1.65 1.28
N 22 22 22 21
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
DOSE GROUP : 1 2 3 4
-----------------------------------------------------------------------------------------------------------------------------------
NO. % NO. % NO. % NO. %
FEMALES EXAMINED AT
SCHEDULED NECROPSY 22 100.0 22 100.0 22 100.0 22 100.0
NONGRAVID 0 0.0 0 0.0 0 0.0 1 4.5
GRAVID 22 100.0 22 100.0 22 100.0 21 95.5
WITH RESORPTIONS ONLY 0 0.0 0 0.0 0 0.0 0 0.0
WITH VIABLE FETUSES 22 100.0 22 100.0 22 100.0 21 100.0

TOTAL FEMALES GRAVID 22 100.0 22 100.0 22 100.0 21 95.5


Early or late resorptions:
no effects observed
Description (incidence and severity):
No treatment related effects
early resorptions: 11, 18, 21 and 26 at 0, 100, 300 and 1000 mg/kg bw
late resorptions: 2, 1, 0 and 1 at 0, 100, 300 and 1000 mg/kg bw

DOSE GROUP : 1 2 3 4

EARLY RESORPTIONS (%)
MEAN 4.5 7.8 7.9 11.2
S.D. 6.50 9.17 16.41 13.30
N 22 22 22 21
LATE RESORPTIONS (%)
MEAN 1.0 0.4 0.0 0.5
S.D. 3.22 1.64 0.00 2.18
N 22 22 22 21
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses in any of the dose group
The number of viable fetuses at 1000 mg/kg bw was reduced compared to controls (88% versus 93% in controls), but this decrease was only slightly outside the historical control values and therefore considered not toxicologically relevant.

DOSE GROUP : 1 2 3 4
VIABLE FETUSES (%)
MEAN 94.6 91.9 92.1 88.3
S.D. 7.64 9.00 16.41 14.16
N 22 22 22 21
DEAD FETUSES (%)
MEAN 0.0 0.0 0.0 0.0
S.D. 0.00 0.00 0.00 0.00
N 22 22 22 21

Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Description (incidence and severity):
1 female at 1000 mg/kg bw was not pregnant
As all females were mated before start of treatment, the test item cannot have had an effect on the initial pregnancy rate.
Details on maternal toxic effects:
none observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
mean fetal body weight: 5.0, 5.2, 5.1 and 5.2 at at 0, 100, 300 and 1000 mg/kg bw

DOSE GROUP : 1 2 3 4
MALE FETAL WEIGHTS (g)
MEAN 5.2 5.3 5.2 5.4
S.D. 0.31 0.35 0.30 0.30
N 22 22 22 21
FEMALE FETAL WEIGHTS (g)
MEAN 4.9 5.0 4.9 5.1
S.D. 0.29 0.29 0.27 0.28
N 22 22 22 21
COMBINED FETAL WEIGHTS (g)
MEAN 5.0 5.2 5.1 5.2
S.D. 0.27 0.29 0.26 0.28
N 22 22 22 21



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
Description (incidence and severity):
mean number of fetuses per litter: 10, 10.5, 10.2 and 9.3 per litter at 0, 100, 300 and 1000 mg/kg bw

The decrease at 1000 mg/kg bw did not reach statistical significance and was only slightly outside the range of the available historical data. Therefore this effects was considered toxicologically irrelevant

DOSE GROUP : 1 2 3 4
VIABLE FETUSES (%)
MEAN 94.6 91.9 92.1 88.3
S.D. 7.64 9.00 16.41 14.16
N 22 22 22 21
DEAD FETUSES (%)
MEAN 0.0 0.0 0.0 0.0
S.D. 0.00 0.00 0.00 0.00
N 22 22 22 21

Changes in sex ratio:
no effects observed
Description (incidence and severity):
44:56, 53:47, 51:49 and 47:53 for the control, 100, 300 and 1000 mg/kg

DOSE GROUP : 1 2 3 4

MALES (%)
MEAN 43.8 53.4 50.7 47.3
S.D. 16.20 14.38 16.86 15.51
N 22 22 22 21

FEMALES (%)
MEAN 56.2 46.6 49.3 52.7
S.D. 16.20 14.38 16.86 15.51
N 22 22 22 21
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
one control fetus: omphalocele (chance finding)

DOSE GROUP: fetuses 1 2 3 4 litters 1 2 3 4
-----------------------------------------------------------------------------------------------------------------------------
NUMBER EXAMINED EXTERNALLY 221 232 225 196 22 22 22 21
TRUNK- OMPHALOCELE 1 0 0 0 1 0 0 0
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Not treatment related
100 mg/kg bw: smal mandible (1), bent limb bone(1), vertebral centra anomaly (1) and sternoschisis (1)
300 mg/kg bw: bent limb bone(4)
1000 mg/kg bw: malpositioned metatarsals (1) and bent limb bone(1)

bent limb done: no relation to dose concentration and as it is also the most common observed malformation in historical controls
other findings: chance findings

DOSE GROUP: fetuses 1 2 3 4 litters 1 2 3 4
-----------------------------------------------------------------------------------------------------------------------------
NUMBER EXAMINED SKELETALLY 110 116 114 100 22 22 22 21
MANDIBLE(S)- ABSENT OR SMALL 0 1 0 0 0 1 0 0
METACARPAL(S) AND/OR METATARSAL(S)-
MALPOSITIONED 0 0 0 1 0 0 0 1
BENT LIMB BONE(S) 1 1 4 1 1 1 3 1
VERTEBRAL CENTRA ANOMALY 0 1 0 0 0 1 0 0
STERNOSCHISIS 0 1 0 0 0 1 0 0

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
one fetus at 100 mg/kg bw: one kidney missing (chance finding)

DOSE GROUP: fetuses 1 2 3 4 litters 1 2 3 4
-----------------------------------------------------------------------------------------------------------------------------
NUMBER EXAMINED VISCERALLY 111 116 111 96 22 22 22 21
KIDNEY(S) AND/OR URETER(S)- ABSENT 0 1 0 0 0 1 0 0
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral variations
DOSE GROUP: fetuses 1 2 3 4 litters 1 2 3 4
-----------------------------------------------------------------------------------------------------------------------------
NUMBER EXAMINED VISCERALLY 111 116 111 96 22 22 22 21
LIVER- APPENDIX 4 2 2 5 4 2 2 5
LIVER- SMALL SUPERNUMERARY LOBE(S) 11 8 9 3 9 8 8 3


Skeletal variations : 84.8%, 78.0%, 84.2% and 79.7% per litter at 0, 100, 300 and 1000 mg/kg bw
DOSE GROUP: fetuses 1 2 3 4 litters 1 2 3 4
-----------------------------------------------------------------------------------------------------------------------------
NUMBER EXAMINED SKELETALLY 110 116 114 100 22 22 22 21
14TH RUDIMENTARY RIB(S) 63 62 57 54 21 21 20 19
PELVIC GIRDLE- CAUDAL SHIFT 4 8 15 7 3 5 7 5
14TH FULL RIB(S) 8 7 16 7 5 6 6 6
REDUCED OSSIFICATION OF THE SKULL 20 18 16 13 13 10 10 7
BENT RIB(S) 22 25 24 18 14 12 11 10
STERNEBRA(E) MALALIGNED (SLIGHT OR MODER) 32 23 32 20 18 14 16 12
METACARPAL(S) AND/OR METATARSAL(S)
UNOSSIFIED 4 5 3 4 4 4 3 2
7TH CERVICAL OSSIFICATION SITE(S) 3 4 8 4 3 4 7 3
7TH CERVICAL FULL RIB(S) 1 2 1 1 1 2 1 1
STERNEBRA(E)- BRANCHED 0 1 0 1 0 1 0 1
VERTEBRAL CENTRA- REDUCED OSSIFICATION 1 1 0 0 1 1 0 0
SKULL- SUPERNUMERARY SITE 0 1 0 0 0 1 0 0
VERTEBRAL ARCHES- REDUCED OSSIFICATION 1 0 0 0 1 0 0 0
STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED 0 0 1 0 0 0 1 0

Details on embryotoxic / teratogenic effects:
no treatment related effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related effects

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
incidental and/or within historical controls

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Any other information on results incl. tables

For detailed results and historical data see attached tables

Maternal data

Dose

0

100

300

1000

Treatment related

Endpoint

 

 

 

 

 

Mortality

No mortality

No

Clinical signs

-Alopecia

 

1/22

 

0/22

 

1/22

 

1/21

 

No

Pregnancy rate

22/22

22/22

22/22

21/22

No

Abortions

0

0

0

0

No

Body weight (gain)

NTRE

No

Body weight (gain) corr. for gravid uterus weight

Not reported

 

Food consumption

NTRE

No

Macroscopy

-Alopecia

 

1/22

 

0/22

 

1/22

 

1/21

 

No

Gravid Uterus weight (g)

68.7

73.1

69.0

65.7

No

Corpora Lutea

12.1

11.6

11.4

11.3

No

Implantation sites

10.6

11.4

11.2

10.6

No

Early resorptions (%)

4.5

7.8

7.9

11.2

No
Not statistically significant and only slightly < historical controls

Late resorptions (%)

1.0

0.4

0

0.5

No

Pre-Implantation loss (mean %)

1.5

0.2

0.2

0.5

No

within historical controls

Post-Implantation loss (mean %)

0.6

0.9

1.0

1.3

No

Viable foetuses (%/mean)

94.6/10

91.9/10.5

92.1/10.2

88.3/9.3

No
Not statistically significant and only slightly < historical controls

Dead foetuses (%)

0

0

0

0

No

NTRE= no treatment related effects

statistically decreased

Fetal data

Dose

0

100

300

1000

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Fetal weight (mean g)

5.0

5.2

5.1

5.2

No

Sex ratio (%)

44

56

53

47

51

49

47

53

No

External findings(% litters affected)

- Omphalocele (no. foetuses)

0.6

1/221

0

0

0

No

Visceral malformations(no foetuses/no litters)

0/0

1/1

0/0

0/0

No

Malformations (no of foetuses):

-Kidneys absent

Variations (no of foetuses):
-Small supernumerary liver lobes/ appendix of the liver

 

 

 

11/4

 

1

 

8/2

 

 

 

9/2

 

 

 

3/5

No

Skeletal malformations(no foetuses/no litters)

1/1

4/4

4/3

2/2

No

Malformations (no foetuses)

- bent limb bones

- malpositioned metatarsals

- small mandibles

- vertebral centra anomaly

- sternoschisis

 

1

 

1

1

1

1

1

 

4

 

1

1

No

Variations (% litters)

84.8

78.0

84.2

79.7

No

*PELVIC GIRDLE- CAUDAL SHIFT,14TH FULL RIB(S), REDUCED OSSIFICATION OF THE SKULL, BENT RIB(S), STERNEBRA(E) MALALIGNED (SLIGHT OR MODERATE), METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED, 7TH CERVICAL OSSIFICATION SITE(S), 7TH CERVICAL FULL RIB(S), STERNEBRA(E)- BRANCHED, VERTEBRAL CENTRA- REDUCED OSSIFICATION, SKULL- SUPERNUMERARY SITE ,VERTEBRAL ARCHES- REDUCED OSSIFICATION AND STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED

1.Formulation Analysis

Accuracy of preparation

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The mean accuracy was 98% for Group 2, 96% for Group 3 and 103% for Group 4.

No test item was detected in the Group 1 formulation.

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). The coefficient of variation was 2.7% for Group 2 and 2.8% for Group 4.

2.Maternal Findings

For further detail on summary data,see attached document with tables.

2.1.Mortality

No mortality occurred in this study.

2.2.Clinical Signs

No toxicologically relevant clinical signs were noted during the observation period. Alopecia was noted for single females of the control, 300 and 1000 mg/kg groups. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

2.3.Body Weights

Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.

2.4.Food Consumption

Food consumption before or after correction for body weight was similar between treated and control animals over the treatment period.

2.5.Macroscopic Examination

Macroscopic examination at necropsy revealed no toxicologically relevant findings.

The only alteration observed was alopecia, noted for three females, confirming the clinical sign observed during the in-life phase.

2.6.Maternal Pregnancy Data

One female at 1000 mg/kg (no. 82) was not pregnant. All other females were pregnant and had litters with viable fetuses.

As all females were mated before start of treatment, the test item cannot have had an effect on the initial

pregnancy rate.

There were no toxicologically relevant effects on the numbers of pregnant females, corpora lutea and implantation sites, or in pre- or post-implantation loss by treatment up to 1000 mg/kg. 

The statistically significantly lower pre-implantation loss at 100 and 300 mg/kg were considered to be caused by a relatively high concurrent control value (11.2% compared to historical control mean value of 6.2%)

3.Fetal Findings

3.1.Litter Size

There were no treatment-related effects on litter size of any group.

Mean litter sizes were 10.0, 10.5, 10.2 and 9.3 fetuses/litter for the control, 100, 300 and 1000 mg/kg groups, respectively.

The percentage of viable fetuses was slightly lower in the 1000 mg/kg group (88.3%), when compared to the control group (94.6%). This was related to the slightly higher percentage of early resorptions at 1000 mg/kg (11.2%), compared to controls (4.5%). As these changes were not statistically significant and only slightly outside the range of the available historical control data, they were not considered to be toxicologically relevant.

Range of historical control data of viable fetuses/litter and early resorptions/litter: 88.9% - 98.4% and 1.6% -

11.1%, respectively.

3.2.Sex Ratio

The male:female ratio was unaffected by treatment up to 1000 mg/kg.

Mean sex ratios (males:females) were 44:56, 53:47, 51:49 and 47:53 for the control, 100, 300 and 1000 mg/kg groups, respectively.

3.3.Fetal Body Weight

There were no effects on fetal body weights (both sexes) noted by treatment up to 1000 mg/kg.

Mean combined (male and female) fetal body weights were 5.0, 5.2, 5.1 and 5.2 gram for the control, 100, 300 and 1000 mg/kg groups, respectively.

4.Fetal Morphological Examinations

The numbers of fetuses (litters) available for morphological examination were 221 (22), 232 (22), 225 (22) and 196 (21) in Groups 1, 2, 3, and 4, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses.

4.1.Exteral Malformations and Variations

There were no treatment related effects on external morphology following treatment up to 1000 mg/kg.

The only external malformation (an omphalocele) observed in this study was noted in control fetus A020-10 and as such was considered a chance finding.

External variations were not seen in any group.

4.2.Visceral Malformations and Variations

There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg.

One visceral malformation was observed, which occurred in fetus A025-12 at 100 mg/kg. This fetus missed one of the two kidneys. As this rare malformation occurred singly, it was considered a chance finding.

The two visceral variations that were noted in this study both involved the liver (small supernumerary liver lobes and appendix of the liver) and as these occurred at low incidences and in the absence of a dose-related incidence, they were not considered to be treatment related.

4.3.Skeletal Malformations and Variations

There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/kg.

Skeletal malformations were observed in 1 (1), 4 (4), 4 (3) and 2 (2) fetuses (litters) in Groups 1, 2, 3 and 4, respectively. The most common malformation was bent limb bones (scapulas and/or humeri) that occurred in one control (A008-02), one Group 2 (A031-11), four Group 3 (A047-11, A056-08, A065-07 and -09) and one Group 4 fetus (A084-09). The group distribution of this malformation did not indicate a relation to dose concentration and as it is also the most common observed malformation in historical controls, it was not considered tobe toxicologically relevant.

Other malformations that were revealed included malpositioned metatarsals noted in Group 4 fetus (A088-03) and small mandibles, vertebral centra anomaly and sternoschisis noted in Group 2(fetus A044-09, A033-03 and A032-05, respectively). Because these occurred singly, they were considered chance findings.

Skeletal variations occurred at an incidence of 84.8%, 78.0%, 84.2% and 79.7% per litter in Groups 1, 2, 3 and 4, respectively. All the ones noted, were not considered to be treatment related as they occurred in the absence of a dose-dependent relationship, infrequently and/or at frequencies that were within the range of available historical control data.

 

Applicant's summary and conclusion

Conclusions:
Twenty-two female rats/group were exposed to the substance during day 6 to 20 post-coitum at 0, 100, 300 and 1000 mg/kg bw by gavage. No effects on maternal mortality, bodyweight (gain), clinical signs, food consumption and macroscopy were found. No effects were reported on maternal develomental parameters like abortions, implantation loss, resorptions and fetal deaths. The litter size as well as the sex ratio, number and weight of the fetuses did not differ between dose groups. No treatment related external, visceral and/or skeletal malformations were observed. The NOAEL for maternal toxicity is 1000 mg/kg bw. The NOAEL for developmental toxicity is 1000 mg/kg bw.
Executive summary:

Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 100, 300 and 1000 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. Gross lesions were collected and fixed from all animals at necropsy. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations.

RESULTS

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings

No maternal toxicity (i.e. mortality, clinical signs of toxicity, toxicologically relevant changes in body weight or food consumption or macroscopic alterations) was observed in the 100, 300 and 1000 mg/kg groups.

Developmental findings

No developmental toxicity (i.e. toxicologically relevant changes in the number of implantation sites, pre- or post-implantation loss, litter size, sex ratio, fetal body weights or fetal morphological alterations) was observed in the 100, 300 and 1000 mg/kg groups.

CONCLUSION

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Levels (NOAEL) for reaction products of tall oil fatty acids with diethylenetriamine and maleic anhydride were established as being 1000 mg/kg.