C,C'-azodi(formamide)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

rabbit

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental start date was 07 Aug 2020.
The in-life phase of the Main study was from 21 Sep 2020 to 06 Oct 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was initiated after final ECHA decision (Decision number: CCH-D-2114461492-49-01/F).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C,C'-azodi(formamide)

EC Number:

204-650-8

EC Name:

C,C'-azodi(formamide)

Cas Number:

123-77-3

Molecular formula:

C2H4N4O2

IUPAC Name:

diazene-1,2-dicarboxamide

Test material form:

solid: particulate/powder

Details on test material:

Batch (Lot) Number: 9J74D
Expiry date: 19 October 2020
Physical Description: Yellowish fine powder
Purity/Composition: 99%
Storage Conditions: At room temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

On 16 Sep 2020 and 18 Sep 2020, time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1-2 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 18-20 weeks old and weighed between 2947 g and 4296 g at the initiation of dosing. A health inspection was performed upon receipt of the animals.

Animal Identification
At study assignment, each animal was identified using a chip.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

At arrival, animals were assigned to groups at random.

Housing
On arrival and following randomization females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The rooms in which the animals were kept was documented in the study records.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study
No., group and animal number.

Environmental Conditions
Target temperatures of 17 to 21°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 18 to 19°C with an actual daily mean relative humidity of 41 to 89%. The values that were outside the targeted range occurred for two non-coherent days with a maximum of 74-91% but were without a noticeable effect on the clinical condition of the animals or on the outcome of the study. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
On the day of arrival, animals received approximately 25 grams of pelleted diet for rabbits
(KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit
AG, Kaiseraugst, Swizerland). During the remainder of the study, the animals received
140-160 grams food per day, except during designated procedures.
In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during
the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Individual animals (in all groups) were observed with a fluctuating food consumption. Apple pieces were provided on various days to stimulate bowel movement by food consumption.

Water
Municipal tap water was freely available to each animal via water bottles/containers. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with shelters (Ebeco,
Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands). Results of analysis for contaminants were provided by the supplier and are on file at the Test Facility. It was considered that there were no known contaminants that would interfere with the objectives of the study.

Veterinary Care
Veterinary care was available throughout the course of the study, and three animals were examined by the veterinary staff as warranted by clinical signs or other changes.
Female No. 44 (125 mg/kg bw/day) was examined on Day 7 post-coitum, as after multiple dosing attempts red fluid was noted on the dosing tube. During auscultation nothing abnormal was noted. The animal was regarded being active and alert and did not have breathing difficulties. Therefore, it was decided to keep this animal on study. Female Nos. 18 (control) and 41 (125 mg/kg bw/day) were examined on Day 14 post-coitum, as after dosing red fluid was noted on the dosing tube and/or breathing difficulties were observed. Both animals were showing labored respiration and were sacrificed in extremis at the advice of the veterinarian.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aqueous

Details on exposure:

Rationale for vehicle
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred
at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis
Dose formulation samples were collected for analysis in Week 1 (21-Sep-2020). The concentration was analysed for all groups and homogeneity analysed for Groups 2 and 4.
The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.
All samples to be analyzed were transferred (at room temperature under normal laboratory
light conditions) to the analytical laboratory at the Test Facility for the same day analysis.
Residual samples were discarded after completion of the sample analysis.

Analytical Method
Analyses were performed using a validated analytical procedure (Test Facility Study
No. 20235609).

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were
within or equal to ±15% for suspensions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of
concentrations was <=10% for suspensions for each group.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and
validation study (Test Facility Study No. 20235609) demonstrated that the test item is stable
in the vehicle when prepared and stored under the same conditions at concentrations
bracketing those used in the present study. Stability data have been retained in the study
records for Test Facility Study No. 20235609.

Results: Accuracy
The concentrations analyzed 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%).
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%).

Details on mating procedure:

Time mated female NZW rabbits were received from Charles River, Chatillon sur Chalaronne, France. The mating procedure is not described in the study report.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum, inclusive.
Female Nos. 15, 29 and 81 of the control, 125 and 500 mg/kg bw/day groups, respectively, were not dosed on Day 10, 11 or 26 post-coitum, respectively as difficulties were noted when inserting the gavage tube. The omission of one day of dosing of a single female over a period of several weeks was considered not to affect the toxicological evaluation.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe. The dosing formulations were stirred continuously during dose administration.

Frequency of treatment:

once daily

Duration of test:

Animals were sacrificed on GD 29.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Justification of Route and Dose Levels
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of the dose range finder (CRL No. 20235617) in an attempt to produce graded responses to the test item.

Examinations

Maternal examinations:

Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Animals showing pain, distress or discomfort which was considered not transient in nature or
was likely to become more severe, were sacrificed for humane reasons.

Clinical Observations
Clinical observations were performed at least once daily, beginning on Day 7 post-coitum and lasting up to the day prior to necropsy. During the dosing period, these observations were performed postdose. Cage debris was examined to detect abortion or premature birth.

Body Weights
Animals were individually weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. Body weights of Day 0 post-coitum (i.e. the day of mating) were collected non-GLP by the supplier.
In order to monitor the health status, Animal Nos. 21, 75, 80, 81 and 87 were weighed on several days from Day 22-28 post-coitum.

Food Consumption
Food consumption was quantitatively measured daily from Day 3 post-coitum onwards.

Water Consumption
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

Terminal Procedures
The following procedures were conducted on 22 females in each group at the scheduled euthanasia day (29 post-coitum): Necropsy; collection of gross lesions; uterus weights. For unscheduled deaths only necropsy and collection of gross lesions was performed.

Unscheduled Deaths
Female Nos. 18 and 21 (control), 41 (125 mg/kg bw/day), 55 (250 mg/kg bw/day) and 81 (500 mg/kg bw/day) were euthanized for humane reasons. These animals were euthanized by intravenous injection of sodium pentobarbital, underwent necropsy, and specified tissues were retained.

Scheduled Euthanasia
Animals surviving until scheduled euthanasia were euthanized by intravenous injection of sodium pentobarbital. No body weight was recorded at necropsy. Scheduled necropsy was conducted on the following days:
Females surviving to planned necropsy: Day 29 post-coitum.
Females with early delivery
(Nos. 74 and 80; 500 mg/kg bw/day):
Within 24 hours of early delivery.

Necropsy
All animals (including animals sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

Organ Weights
The (gravid) uterus was weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in poor condition or in extremis.

Ovaries and uterine content:

Each ovary and uterine horn of all animals was dissected and examined in case of pregnancy as quickly as possible to determine in pregnant females only:
- The number of corpora lutea.
- The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy or
that delivered early).
- The number of implantation sites.
- The number and distribution of live and dead fetuses.
- The number and distribution of early and late resorptions.
For animals sacrificed before planned necropsy or that delivered before the day of scheduled
necropsy, findings were reported in the individual data tables only. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

Blood sampling:

Blood sampling was not conducted.

Fetal examinations:

Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. As for Fetus No. 5 of Litter No. 39 (125 mg/kg bw/day) this was not possible due to a malformation, this fetus was euthanized by intrascapular injection of sodium pentobarbital. Recognizable live fetuses of animals sacrificed before planned necropsy were euthanized by oral administration of sodium pentobarbital. As pups of females that started to deliver were already dead, these pups did not needed to be euthanized.

Fetal Examinations (unscheduled)
For recognizable fetuses in development of females sacrificed before planned necropsy or that delivered before the day of scheduled necropsy, a gross external examination was performed. Late resorptions and recognizable fetuses with abnormalities were fixed in 10% buffered formalin.

Fetal Examinations (scheduled)
Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined. Nonviable fetus 8-R5 (control) was examined and weighed, as the degree of autolysis was minimal or absent.

For late resorptions a gross external examination was performed. Late resorption (Fetus No. 6 of Dam No. 1; control) was observed with malformations and was therefore fixed in 10% buffered formalin.

Visceral Examinations
All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice. All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson.
Subsequently, the skeletal examination was done on all fetuses from all groups. All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. Evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed
appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by
dataset.
All statistical tests were conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and were reported at the 1% or 5% levels.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric/Non-Parametric
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not
significant or the Kruskal-Wallis test if it was significant. If the overall F-test or
Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted
using Dunnett’s or Dunn’s test, respectively.

The following variables were assessed using this technique: Body weight; body weight gains; food consumption; gravid uterine weight and corrected maternal body weights; litter observations.

Non-Parametric
The groups were compared using an overall Kruskal-Wallis test. The following variables were assessed using this technique: Ovarian and Uterine examinations; Litter % of Fetuses with
Gross/External/Visceral/Skeletal Abnormalities.

Incidence
A Fisher’s exact test was used to conduct pairwise group comparisons of interest: Parental indices and mortality.

ANCOVA
The data corresponding to a response variable of interest and to a related covariate were
submitted to an analysis of covariance (ANCOVA), including only groups with at least three
non-missing paired values and if found to be significant, then pairwise comparisons were
conducted using Dunnett’s test.

Indices:

Maternal Variables
Body Weight Gains: Calculated for the following intervals: Days 7 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27, 27 to 29 and 7 to 29 post-coitum.
Corrected Body Weight Gain: Body weight on Day 29 post-coitum - body weight on Day 7 post-coitum - gravid uterus weight.
Overall Food Consumption: Calculated between each scheduled interval
(individual data only) and as specified above for bodyweight gains. Summarization and statistical
analysis intervals will reflect the same intervals as the bodyweight gains.

Pregnancy Rate (%): No. of pregnant females/ No. of mated females x 100

Litter Variables

Sex Ratio (% males): No. male fetuses/Total no. of fetuses x 100

Pre-Implantation Loss (%): (No. of corpora lutea – no. of implants)/No. of corpora lutea x 100

Post Implantation Loss (%): (No. of implants – no. of live fetuses)/No. of implants x 100

Litter % of Fetuses with Abnormalities: No. of fetuses in litter with a given finding/No. of fetuses in litter examined x 100

Historical control data:

No historical control data was provided.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Erected fur was observed on 1-2 days for three animals (one at 250 and two at
500 mg/kg bw/day). For one of the high dose animals, thin appearance was noted on the same
days. As these findings were in line with the findings observed for females sacrificed at the
high dose level, this was considered to be test item related.
Decreased fecal production has been observed in 13/22, 9/22, 18/22 females and 18/22
females of the control, 125, 250 and 500 mg/kg bw/day groups, respectively. In the absence
of a dose response, this was considered to be unrelated to treatment with the test item.
Red discolored discharge from the vagina was observed on Day 18 post-coitum for Female
No. 45 (250 mg/kg bw/day), this was considered an incidental finding.
Any other clinical signs noted during the treatment period occurred within the range of
background findings to be expected for rabbits of this age and strain which are housed and
treated under the conditions in this study and did not show any apparent dose-related trend. At
the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

In total seven females were sacrificed in extremis, of which three deaths at 500 mg/kg bw/day
were considered to be related with treatment with the test item. All females were pregnant at
the time of early sacrifice.
Female Nos. 74 and 80 (500 mg/kg bw/day) were sacrificed on Days 25 and 27 post-coitum,
respectively, after they started to deliver their offspring. Both females were observed with
erected fur, body weight loss (up to 10% compared to their maximum body weight) and near
absent food consumption (nine and ten consecutive days, respectively) during the period prior
to delivery. At necropsy, Female No. 74 was observed with ectopic splenic tissue and wateryclear
cysts on both oviducts, which were considered to be unrelated to treatment with the test
item. Female No. 80 was observed with prominent lobular architecture of the liver. The
delivery of their offspring was most likely caused by the poor general condition of the
animals and was therefore considered to be an indirect effect of the test item.
Female No. 81 (500 mg/kg bw/day) was sacrificed in extremis on Day 26 post-coitum, as it
was observed with absent food consumption for 11 consecutive days and was observed with
erected fur and being thin on Day 26 post-coitum. Body weight loss (up to 4% compared to
its maximum body weight) was observed on Days 18 and 21 post-coitum, with a slight
recovery thereafter. Necropsy revealed ectopic splenic tissue, which was considered to be
unrelated to the test item.
Three animals were sacrificed in extremis following complications of the gavage procedure: Female Nos. 18 (control group) and 41 (125 mg/kg
bw/day) were sacrificed after veterinarian consultation on Day 14 post-coitum, and Female
No. 55 (250 mg/kg bw/day) was sacrificed directly after dosing.
Female No. 21 (control) was sacrificed in extremis on Day 24 post-coitum, as it was observed
with absent food consumption for ten consecutive days.

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.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Overall, mean food consumption was similar to the control level over the study period.
However, mean food consumption at 500 mg/kg bw/day was slightly lower compared to
control between Days 15 and 24 post-coitum but no statistical significance was obtained. In
the period thereafter, food consumption appears to restore to values comparable to control
females, but this is most likely caused by the sacrifice of Female Nos. 74, 80 and 81.
Any statistically significant changes in food consumption were considered to be unrelated to
treatment given the direction of change and as no trend was apparent regarding dose.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment related effects were observed on the gravid uterine weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dark-brown or dark-red foci of the lungs were noted in 3/19 animals at 500 mg/kg bw/day at
scheduled necropsy. As these tissues were not microscopically examined by a pathologist, a
test item relationship could not be excluded.
Other findings that were noted among control and/or treated animals were considered to be of
no toxicological significance, as they remained within the range of biological variation for
rabbits of this age and strain and/or as no trend was apparent regarding dose.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

In total, 20, 21, 21 and 19 females in the control, 125, 250 and 500 mg/kg bw/day groups,
respectively, survived until scheduled necropsy, of which eight females in total (Female
Nos. 9, 12 and 14 (control), Nos. 26, 30, 40 and 43 (125 mg/kg bw/day) and No. 73
(500 mg/kg bw/day) were not pregnant.
Female Nos. 74 and 80 (500 mg/kg bw/day) started to deliver their offspring prior to
scheduled necropsy (Day 25 and 27 post-coitum, respectively). The remaining females were
pregnant and had litters with viable fetuses.
As a result, 17, 17, 21 and 18 females in the control, 125, 250 and 500 mg/kg bw/day groups,
respectively, were available for evaluation.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites, pre- and post-implantation loss and early
and late resorptions in the control and test groups were similar and in the range of normal
biological variation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites, pre- and post-implantation loss and early
and late resorptions in the control and test groups were similar and in the range of normal
biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites, pre- and post-implantation loss and early
and late resorptions in the control and test groups were similar and in the range of normal
biological variation.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

Female Nos. 74 and 80 (500 mg/kg bw/day) started to deliver their offspring prior to
scheduled necropsy (Day 25 and 27 post-coitum, respectively). Both females were observed with
erected fur, body weight loss (up to 10% compared to their maximum body weight) and near
absent food consumption (nine and ten consecutive days, respectively) during the period prior
to delivery. The delivery of their offspring was most likely caused by the poor general condition of the animals and was therefore considered to be an indirect effect of the test item. The remaining females were pregnant and had litters with viable fetuses.

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

In total seven females were sacrificed in extremis, of which three deaths at 500 mg/kg bw/day
were considered to be related with treatment with the test item. All females were pregnant at
the time of early sacrifice.
Female Nos. 74 and 80 (500 mg/kg bw/day) were sacrificed on Days 25 and 27 post-coitum,
respectively, after they started to deliver their offspring. Both females were observed with
erected fur, body weight loss (up to 10% compared to their maximum body weight) and near
absent food consumption (nine and ten consecutive days, respectively) during the period prior
to delivery. At necropsy, Female No. 74 was observed with ectopic splenic tissue and wateryclear
cysts on both oviducts, which were considered to be unrelated to treatment with the test
item. Female No. 80 was observed with prominent lobular architecture of the liver. The
delivery of their offspring was most likely caused by the poor general condition of the
animals and was therefore considered to be an indirect effect of the test item.
Female No. 81 (500 mg/kg bw/day) was sacrificed in extremis on Day 26 post-coitum, as it
was observed with absent food consumption for 11 consecutive days and was observed with
erected fur and being thin on Day 26 post-coitum. Body weight loss (up to 4% compared to
its maximum body weight) was observed on Days 18 and 21 post-coitum, with a slight
recovery thereafter. Necropsy revealed ectopic splenic tissue, which was considered to be
unrelated to the test item.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

effects on pregnancy duration

food consumption and compound intake

mortality

Maternal abnormalities

Key result

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on fetal body weights (both sexes) noted by treatment
up to 500 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 500 mg/kg bw/day.
One fetus at 125 mg/kg bw/day, and two fetuses (from different nests) at 500 mg/kg bw/day were scored as sex undetermined, as it was not traceable from study data what the internal sex of these fetuses was.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related external malformations or variations were noted up to 500 mg/kg bw/day.
External malformations were observed in four live fetuses in four different affected litters at
125 mg/kg bw/day and in one 500 mg/kg bw/day fetus, but not in control fetuses and at
250 mg/kg bw/day.
The number of affected fetuses at 125 mg/kg bw/day was statistically significantly increased,
but the malformations were diverse in nature and as they occurred at the low-dose level they
were considered chance findings. The high-dose fetus had short limbs that was considered
spontaneous in origin due to single occurrence. Besides fetal external malformations, there was one late resorption in the control group (1-L6) with a hyperflexed forepaw.
There were no external variations noted in any groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related skeletal malformations or variations were observed up to 500 mg/kg
bw/day.
There were 4 (4), 12 (5), 3 (2) and 5 (4) fetuses (litters) with skeletal malformations in the
control, 125, 250 and 500 mg/kg bw/day groups, respectively.
For two fetuses at 125 mg/kg bw/day and one fetus at 500 mg/kg bw/day, the skeletal malformations were associated with external malformations.
Of the other malformations, supernumerary lumbar vertebra occurred most frequently,
namely in 4 (4), 8 (2), 2 (2) and 2 (2) fetuses (litters) of the control, 125, 250 and
500 mg/kg bw/day groups, respectively. However, this group distribution does not indicate
any test item-relationship.
Remaining malformations affected thoracic vertebrae and/or ribs and occurred singly and
therefore were considered chance findings.
All skeletal variations occurred in the absence of a dose-related incidence trend and/or
infrequently. Therefore, they were considered not test item related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related visceral malformations or variations were observed up to
500 mg/kg bw/day.
Visceral malformations affected two control fetuses and one fetus each in the 125 and
500 mg/kg bw/day groups.
The high-dose fetus had a malpositioned adrenal gland and the fetuses in the control group
and at 125 mg/kg bw/day had various cardiovascular abnormalities of which one (a control
fetus) also had fused lung lobes. The incidental occurrence and group distribution of
these malformations did not suggest a test item-related effect and therefore they were
considered to have occurred by chance.
All variations were seen in single fetuses or observed in groups that received the test item
with incidences comparable to the control group.

Details on embryotoxic / teratogenic effects:

No test item-related changes were noted in any of the developmental parameters investigated
in this study (i.e. litter size, pre- and post-implantation loss, sex ratio, fetal body weights,
external, visceral and skeletal malformations and developmental variations).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed (tested up to a dose level resulting in significant maternal toxicity)

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of a prenatal developmental toxicity study performed according to OECD/EC guideline and GLP principles, the maternal NOAEL was established at 250 mg/kg bw/day, based on reduced body weights and food intake resulting in mortality or early delivery at 500 mg/kg bw/day. The developmental NOAEL was concluded to be at least 500 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study was performed with the registered substance according to OECD/EC guideline and GLP principles. The objectives of this study were to determine the potential of Azodicarbonamide Unifoam AZ VI-50 to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 7 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 125, 250, 500 mg/kg bw/day based on the results of a Dose Range Finder Study (CRL study 20235617). The study design was as follows:

Group No	Test Item Identification	Dose Level (mg/kg bw/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Females
1		0(Control)	5	0	22
2	Azodicarbonamide Unifoam AZ VI-50	125	5	25	22
3		250	5	50	22
4		500	5	100	22

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.
The following parameters and endpoints were evaluated in this study for the F0-generation:
mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.
In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.
Formulation analyses confirmed that formulations of test item in 1% aqueous carboxymethyl cellulose were prepared accurately and homogenously.
In total, seven females were sacrificed before scheduled necropsy, of which three females were sacrificed in extremis as a result of treatment with the test item. Of these three females, two started to deliver their offspring prior to scheduled necropsy, most likely as an indirect effect of their poor general condition as females were observed with erected fur, body weight loss and near absent food consumption during the period prior to delivery. The third female was sacrificed in extremis as it was observed with absent food consumption for 11 consecutive days and was observed with erected fur and being thin on the day of sacrifice.
The early sacrifice of the other four females were considered to be unrelated to treatment with the test item and were a result of complications of the gavage procedure or absent food consumption.
Dark-brown or dark-red foci of the lungs were noted in 3/19 animals at 500 mg/kg bw/day at scheduled necropsy. As these tissues were not microscopically examined by a pathologist, a test item relationship could not be excluded.
No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, pre- and post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

Based on these results, the maternal NOAEL was established to be 250 mg/kg bw/day, and the developmental NOAEL was concluded to be at least 500 mg/kg bw/day.