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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:
19 OCTOBER 2012 to 24 JANUARY 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to current test guidelines and GLP compliant
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): AZDN
- Physical state: White powder
- Analytical purity: 99%
- Purity test date: 08 August 2012
- Lot/batch No.: 7582
- Expiration date of the lot/batch: 31 March 2013
- Stability under test conditions: Suspensions of 0.1 and 15 mg/mL were found to be stable for up to 11 days at 2 to 8C (study no. 8266542).
- Storage condition of test material: 2 to 8C in the dark

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Age at study initiation: 9-10 weeks old at time of mating
- Weight at study initiation: 200.1 to 271.8 g at time of mating
- Fasting period before study: No
- Housing: Animals housed singly in cages that conform with the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989). Suitable wood bedding provided weekly to each cage. Wooden Aspen chew blocks and sizzle nest provided as environmental enrichment.
- Diet (e.g. ad libitum): SQC Rat and Mouse Breeder Diet No 3, Expanded (Special Diets Services Ltd, Witham, UK) ad libitum
- Water (e.g. ad libitum): Mains water ad libitum
- Acclimation period: No. Female animals delivered to the testing laboratory on Day 3 of gestation and examined on receipt. All animals found to be in good health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24C
- Humidity (%): 45 to 65%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 19 October 2012 to 08 November 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared on three occasions. The test substance was formulated as a suspension in corn oil following dispensary SOPs and the formulation method 8266543_O_01D.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil selected due to stability of test substance in the vehicle, and use in previous study R-95-007.
- Concentration in vehicle: Not specified
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity previously assessed at 0.1 and 15 mg/mL in study 8266542.
CONCENTRATION VERIFICATION: Samples (three random aliquots from test substance formulations; two random aliquots from control formulations) prepared for use on the first and last day of dosing were taken for analysis of achieved concentration.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Mating was performed overnight at the supplier's laboratory and confirmed by the presence of a vaginal plug in situ.
Duration of treatment / exposure:
The test and control substances were administered orally, by gavage, to mated female rats daily from Day 6 to Day 19 of gestation.
Frequency of treatment:
Daily
Duration of test:
The females were maintained to Day 20 of gestation when they were killed and their uterine contents examined.
No. of animals per sex per dose:
22 females per dose concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level of 20 mg/kg/day was selected as this was expected to elicit mild maternal toxicity and was based on the range-finding study 8266558, where a dose level of 25 mg/kg/day elicited maternal toxicity in the form of slight body weight loss and reduced food consumption. The intermediate dose level of 5 mg/kg/day was selected as this is the geometric mean of the high and low dose levels. The low dose level of 1 mg/kg/day was selected as this was expected to be a no observed effect level (NOEL).

- Rationale for animal assignment (if not random): On Day 3 of gestation the animals were assigned to treatment groups using a randomisation procedure based on body weight and day of mating.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Beginning and end of the working day for signs of ill health and overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination on the days of body weight recording.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each female was recorded on Days 3, 6, 7, 8, 9, 12, 15, 17, 19 and 20 of gestation.

FOOD CONSUMPTION):
Individual food consumption was recorded for Days 3 to 5, 6, 7, 8, 9 to 11, 12 to 14, 15 to 16 and 17 to 18 and 19 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: Ovaries and uteri
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
Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those which appeared to have died shortly before necropsy.
Fetal examinations:
Live foetuses were killed by a subcutaneous injection of sodium pentobarbitone.
Individual foetal and placental weights were recorded and foetuses were examined externally and sexed. Approximately one half of the foetuses in each litter, selected by systematic sampling, were examined for visceral abnormalities by microdissection. They were then eviscerated and the carcasses processed to stain the ossified skeleton by the Alizarin technique and cartilage processed to stain using Alcian Blue. The skeletons were examined, preserved and stored in glycerol/propylene glycol.
The remaining foetuses were placed in Bouin's solution for at least two weeks to allow fixation and partial decalcification. At examination, the head was removed by a cut through the mouth, pharynx and back of the head and coronal sections of the head were examined. The remaining portion of the foetus was examined by dissection and was preserved, with the head sections, in 10% neutral buffered formalin and stored in plastic vials.
The dissection of the foetuses and the examination of the stained skeletons were performed using low power binocular magnification.
Foetal abnormalities were classified as malformations (rare and/or potentially lethal defects) and variations (commonly occurring non lethal abnormalities).
Statistics:
The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3 and 4) were compared.
Statistical methods included one-way analysis of variance (ANOVA), Levene's test, Dunnett's test, Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test, the Wilcoxon rank sum test, analysis of covariance (ANCOVA), Cochran-Armitage test and Fisher's exact test.
Indices:
Not applicable
Historical control data:
Not applicable

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal receiving 1 mg/kg/day (number 23) died on Day 7 of gestation and one animal receiving 5 mg/kg/day (number 62) died on Day 20 of gestation. Findings for these animals were consistent with dosing accidents including thoracic cavity containing abnormal liquid contents.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slight body weight loss was seen in animals receiving 5 and 20 mg/kg/day from Days 6 to 8 of gestation inclusive. Statistically significant reduced body weight gain from Days 7 to 8 of gestation at 5 and 20 mg/kg/day (79% and 173% less, or P<0.05 and P<0.001 respectively). Percentage body weight change (corrected) on Day 20 of gestation was lower than control at 5 and 20 mg/kg/day (12% and 29% lower respectively). These were considered to be effects of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In animals receiving 5 and 20 mg/kg/day there was a statistically significant treatment-related reduction in food intake from Days 6 to 8 of gestation inclusive (between 5% and 47% less, or P<0.05 for both), with recovery seen afterwards. This was an effect of treatment.
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
Gross pathological findings:
effects observed, non-treatment-related
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):
There were 22, 22, 22 and 22 pregnancies leading to 22, 21, 21 and 22 litters in animals receiving 0, 1, 5 and 20 mg/kg/day respectively. The mean number of corpora lutea, the mean incidence of pre- and post-implantation loss and mean litter size were all unaffected by treatment.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Sex ratio, mean litter weight, mean placental weight and mean foetal weight all showed no effect of treatment.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Morbidity and mortality:
One animal receiving 1 mg/kg/day (number 23) died on Day 7 of gestation and one animal receiving 5 mg/kg/day (number 62) died on Day 20 of gestation. Findings for these animals were consistent with dosing accidents including thoracic cavity containing abnormal liquid contents.
There were no unscheduled treatment-related deaths.

Clinical signs:
There were no significant, treatment-related clinical signs recorded.

Post-dosing observations:
On the first dosing the post-dosing observation mouth rubbing was seen in two animals receiving 1 mg/kg/day and three animals receiving 5 mg/kg/day. During the rest of the dose period mouth rubbing was seen on most days in up to six animals receiving 20 mg/kg/day. This commonly recorded observation is considered to be a result of taste-aversion rather than systemic toxicity.

Body weight:
Slight body weight loss was seen in animals receiving 5 and 20 mg/kg/day from Days 6 to 8 of gestation inclusive. Statistically significant reduced body weight gain from Days 7 to 8 of gestation at 5 and 20 mg/kg/day (79% and 173% less, or P<0.05 and P<0.001 respectively). Percentage body weight change (corrected) on Day 20 of gestation was lower than control at 5 and 20 mg/kg/day (12% and 29% lower respectively). These were considered to be effects of treatment.

Food consumption:
In animals receiving 5 and 20 mg/kg/day there was a statistically significant treatment-related reduction in food intake from Days 6 to 8 of gestation inclusive (between 5% and 47% less, or P<0.05 for both), with recovery seen afterwards. This was an effect of treatment.

Effect levels (maternal animals)

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

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were noted in three foetuses from three litters in the control group (one external/visceral, two skeletal), four foetuses from three litters at 1 mg/kg/day (skeletal), five foetuses from two litters at 5 mg/kg/day (skeletal), and nine foetuses from eight litters at 20 mg/kg/day (three external/visceral, six skeletal). The incidence and intergroup distribution of these foetal malformations and variations do not indicate an adverse effect of treatment. Findings which on occasion achieved statistical significance generally fell within the background range (abnormal shaped head, thymus cervical remnant, metacarpal 5 unossified), or were commonly seen findings which did not show a clear treatment-related increase across the groups and/or were not considered to be biologically significant (kidney cavitation increased, ureter distended – fluid contents).
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Uterine/implantation data:
There were 22, 22, 22 and 22 pregnant females leading to 22, 21, 21 and 22 litters in animals receiving 0, 1, 5 and 20 mg/kg/day respectively. The mean number of corpora lutea, the mean incidence of pre- and post-implantation loss and mean litter size were all unaffected by treatment.

Foetal data:
Sex ratio, mean litter weight, mean placental weight and mean foetal weight all showed no effect of treatment.

Foetal defect data:
Malformations were noted in three foetuses from three litters in the control group (one external/visceral, two skeletal), four foetuses from three litters at 1 mg/kg/day (skeletal), five foetuses from two litters at 5 mg/kg/day (skeletal), and nine foetuses from eight litters at 20 mg/kg/day (three external/visceral, six skeletal). The incidence and intergroup distribution of these foetal malformations and variations do not indicate an adverse effect of treatment. Findings which on occasion achieved statistical significance generally fell within the background range (abnormal shaped head, thymus cervical remnant, metacarpal 5 unossified), or were commonly seen findings which did not show a clear treatment-related increase across the groups and/or were not considered to be biologically significant (kidney cavitation increased, ureter distended – fluid contents) (see attached tables).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 20 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effect

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Summary of Foetal Defects

Defect

Mean incidence – number of foetuses (mean percentage of foetuses)

Background Incidence minimum %, maximum %

 

Group

 

1

2

3

4

External/visceral findings

Malformations

Aortic arch, retro-oesophageal

-

-

-

1 (0.3)

 

Eye, anterior chamber, internal haemorrhage

-

-

-

1 (0.3)

 

Eyes, one absent

-

-

-

1 (0.5)

 

Eyes, severely reduced in size

1 (0.4)

-

-

1 (0.5)

 

Variations

 

 

 

 

 

Head, abnormal shape, domed

2 (0.9)

-

2 (0.7)

9 (3.4)DR*

0.7, 4.4

Kidney, cavitation increased

6 (3.0)

5 (1.7)

4 (1.7)

18 (10.7)*

0.8, 4.9

Thymus, cervical remnant

1 (0.4)

6 (2.1)

7 (2.6)

11 (4.0)*

0.0, 11.2

Ureter, distended – fluid contents

10 (4.6)

6 (2.1)

8 (3.5)

18 (10.8)

1.9, 6.6

Skeletal findings

Malformations

Forelimbs, severely shortened and bent with bent scapulae

-

-

-

1 (0.9)

 

Ribs, ossification interrupted

-

1 (0.8)

-

-

 

Vertebral thoracic centrum, cleft cartilaginous centres

2 (1.3)

1 (0.7)

2 (1.4)

2 (1.6)

 

Vertebral cervical arches, additional cartilaginous ventral plate

-

1 (0.6)

-

-

 

Variations

 

 

 

 

 

Metacarpal 5 , unossified

26 (21.7)

14 (11.6)

38 (30.2)

38 (26.7)*

17.1, 45.2

 

 

 

 

 

 

* P<0.05

DR = significant dose response test

Applicant's summary and conclusion

Conclusions:
In conclusion, administration of AZDN to pregnant rats from Days 6 to 19 of gestation at dose levels of 1 ,5 and 20 mg/kg/day resulted in maternal toxicity from 5 mg/kg/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on foetal development were observed.
At 1 mg/kg/day, no adverse effects were observed in both parental and foetal development.
There was no evidence of embryotoxicity at any dose level tested.
The maternal no observed adverse-effect level (NOAEL) was 1 mg/kg/day based on a significant decrease in maternal body weight gain and food consumption.
The embryo-foetal NOAEL was 20 mg/kg/day.
Executive summary:

The objective of the study was to determine the effects of the test substance, AZDN(2,2’-dimethyl-2,2’azodipropionitrile), on the embryonic and foetal development of the rat.

The test item and control substance (corn oil) were administered orally, by oral gavage, to mated female rats daily from Day 6 to Day 19 of gestation, inclusive. The animals were dosed in ascending group order. The females were maintained to Day 20 of gestation when they were killed and their uterine contents examined.

Groups of rats of the Crl:CD(SD) strain were dosed as follows:

Group Number

Description

Dose level (mg/kg/day)

Number of animals in group

1

Control

0

22

2

Low

1

22

3

Intermediate

5

22

4

High

20

22

 

Two females one from each of the low and intermediate dose groups died on Days 7 and 20 of gestation respectively due to dosing accidents. Findings for these animals were consistent with dosing accidents including thoracic cavity containing abnormal liquid contents. There were no treatment-related deaths, clinical signs, or macroscopic necropsy findings.

The minor post-dosing observation mouth rubbing was seen in all treated groups with frequency of observation increasing with increasing dose.

Treatment-related reduced food intake (between 5% and 47% less, or P<0.05) for both 5 and 20 mg/kg/day and a consequent slight body weight loss (79% and 173% less, or P<0.05 and P<0.001 respectively) were seen from Days 6 to 8 of gestation inclusive.

Mean uterine/implantation and mean foetal data showed no adverse effect of treatment. There was no overall increase in the mean incidence or inter-group distribution of external, visceral or skeletal foetal variations or malformations.

In conclusion, administration of AZDN to pregnant rats from Days 6 to 19 of gestation at dose levels of 1 ,5 and 20 mg/kg/day resulted in maternal toxicity from 5 mg/kg/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on foetal development were observed.

At 1 mg/kg/day, no adverse effects were observed in both parental and foetal development.

There was no evidence of embryotoxicity at any dose level tested.

The maternal no-observed-adverse-effect-level (NOAEL) was 1 mg/kg/day based on a significant decrease in maternal body weight gain and food consumption from 5 mg/kg/day upwards.

The embryo-foetal NOAEL was 20 mg/kg/day.