C,C'-azodi(formamide)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 to 15 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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

Details on test material:

Description: Yellowish fine powder
Lot/Batch No.: 9J74D
Purity: 99%
CAS No.: Cas no. 123-77-3
Stability of test compound: Batch expiry date 07 April 2021

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at dosing: 6 wks
Weight at dosing: males: 123-162 g; females: 106-141 g
Source: Charles River Deutschland, Sulzfeld, Germany
Acclimation period: 12 days
Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany ad libitum
Water: Municipal water, ad libitum
Housing: Polycarbonate cages (Makrolon type IV, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group together.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
The room in which the animals were kept was documented in the study records. Cages were arranged on the racks according to a Latin-square model. Cage Identification: Color-coded cage card indicating at least Test Facility Study No., group, animal identification number(s).

Environmental conditions:
Temperature: 21 ±3 °C
Humidity: 55 ± 15%
Air changes: ca. 10/h
Photoperiod: 12 hour light/dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aqueous

Details on oral exposure:

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 weekly as a suspension, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred
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 test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.
Dose formulation samples were collected for analysis from the dosing container in week 1, 6 and 13: Concentration- all groups 2x approx. 500mg; Homegeneity- groups 2 and 4 2 x approx. 500mg (top, middle, bottom).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analysis was performed using a validated analytical procedure (Test Facility Study No. 20235609).
Concentration and Homogeneity Analysis
Storage Conditions: Temperature set to maintain 18-22°C.
Acceptance Criteria: For concentration: mean sample concentration results within or equal to ± 15% of theoretical concentration.
For homogeneity, relative standard deviation (RSD) of concentrations of
= 10% 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.

Outcome
The concentrations analyzed in the formulations of Groups 2-4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 6 because the samples had not been diluted in first instance. After dilution and re-analyzation no test item was detected.
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation = 10%).

Duration of treatment / exposure:

7 days a week for a minimum of 13 weeks.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 (ten)

Control animals:

yes, concurrent vehicle

Details on study design:

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 results of a 14-day repeated dose toxicity study with oral exposure of Azodicarbonamide Unifoam AZ VI-50 in rats, Test Facility Reference No. 20235611), and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Examinations

Observations and examinations performed and frequency:

Observations: Animals were inspected at least once daily for signs of toxicity and twice daily for mortality. Detailed clinical examinations were conducted once before administration (on the day before the administration start day) and once a week during the administration period.
Arena observations were conducted once before first dosing and weekly thereafter.
Body weights: Animals were weighed at initiation of dosing and weekly during administration and at study termination.

Food consumption: Determined by weighing food supplied and food that remained were calculated as time-weighted averages from food consumption and body weight gain data.

Water consumption: Monitored by visual inspection of water bottles

Ophthalmological examination: Eyes were examined before administration and in wk 13 on all control and high dose group animals.

Neurological functional examinations: Once during the Dosing Period. The first 5 animals per sex per group during Weeks 12-13. These tests were performed after clinical
observations.
Procedure: The following tests were performed:
• hearing ability, pupillary reflex and static righting reflex (score 0= normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength were recorded as the mean of three measurements.
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system).

Haematology and clinical chemistry: Conducted on days 91-93. Animals were fasted overnight prior to blood sampling.
Haematology: red blood cell parameters (haematocrit (commonly termed PCV), haemoglobin concentration (Hb), mean haemoglobin concentration (MHC), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), red blood cell distribution width (RDW), erythrocyte count, platelet count, reticulocyte count), white blood cell parameters (total and differential (neutrophils, lymphocytes, eosinophils, basophils, monocytes) leukocyte count), coagulation parameters (activated partial thromboplastin time (APTT), prothrombin time (PT)).
Clinical chemistry: electrolytes (sodium, potassium, calcium, chloride, inorganic phosphorus), kidney function test (creatinine, urea), glucose, liver function tests (albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T.Bili), total protein (TP), lipid profile (total triglyceride, total cholesterol, HDL & LDL cholesterol), T3, T4, TSH.
Hormone profile: FSH, LH, estradiol, testosterone.

Urinalysis: Not conducted

Sacrifice and pathology:

Organ weights: Adrenal glands(a), pituitary gland, brain, epididymides(a), prostate gland, seminal vesicle(a), heart, kidney(a), liver, ovary(a), spleen, thymus, thyroid, testis(a), uterus/cervix.
(a) Paired organ weight

Sacrifice and pathology: Conducted on day 91-93. Gross pathological examination was performed on all animals and included examination of the external surface, all orifices and associated tissues.
The following tissues were preserved in 10% neutral buffered formalin for subsequent histopathological examination and performed on control and high dose group animals.
Animal identification (b;) Artery, aorta; Body cavity, nasal(b); Bone, femur(b); Bone marrow; Bone, sternum; Brain; Cervix; Epididymis(a); Esophagus; Eye(a); Gland, adrenal; Gland, clitoral(b); Gland, harderian(a,b); Gland, lacrimal(b); Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, preputial(b); Gland, prostate; Gland, salivary(b); Gland, seminal vesicle; Gland, thyroid; Large intestine, cecum; Large intestine, colon; Large intestine, rectum; Larynx(b); Liver; Lung; Lymph node, mandibular; Lymph node, mesenteric; Muscle, skeletal; Nerve, optic; Nerve, sciatic; Nerve, tibial(b); Ovary; Pancreas; Skin; Small intestine, duodenum; Small intestine, ileum; Small intestine, jejunum; Spinal cord; Spleen; Stomach; Testis(a); Thymus; Tongue(b); Trachea; Urinary bladder; Uterus; Vagina.
(a) Preserved in Modified Davidson’s fixative prepared at Charles River Den Bosch using Formaldehyde 24 %, Ethanol, Acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA).
(b) not examined for histopathology

Neurohisto-pathology: Not conducted

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. Pairwise comparisons were only conducted between the control and each dose group.
The following parameters were assessed for homogeneity of group varinaces using Levene’s Test: Body weight, body weight gains, food consumption, coagulation variables, clinical chemistry variables, hormone variables, functional observation battery quantitative variables, organ weights and relative organ weights.
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.
A Fisher’s exact test was used to conduct pairwise group comparisons of interest for the functional observation battery qualitative variables.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted in males and females at 100 and 300 mg/kg/day. Clinical signs observed in the animal found dead (100 mg/kg/day) are listed below.
At 1000 mg/kg/day, one male and one female showed erected fur between Days 72 and 78. In addition, three females showed hunched posture between Days 72 and 77. At the incidence observed and based on the relatively short duration and transient nature of these findings, they were considered not toxicologically relevant.
Any other clinical signs (i.e. scabs and tail bent) noted during the dosing period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period, except for one female treated at
100 mg/kg/day (Female No. 59), that was found dead on Day 75 of dosing. Clinical signs observed consisted of hunched posture, erected fur and thin appearance between Days 72 to 74 and body weight loss (8%) was observed between Days 64 and 71.
Macroscopic evaluation showed accumulation of a dark opaque, red fluid in the thoracic cavity, adhesion of the lung to the diaphragm and accumulation of a brown, tan yellow material in the lungs. In addition, histopathological assessment showed an abcess containing plant material in the lung and inflammation of the lung pleura. Based on the microscopic findings the cause of death was considered to be related to the gavage procedure and was not directly test item related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test item.
Any statistically significant changes in body weight gain in males and females were considered to be unrelated to treatment with the test item, since no trend was apparent regarding dose and/or time.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No effects on food consumption were observed in males up to 1000 mg/kg/day.
In all female test item-treated groups a slight decrease in food consumption was observed starting on Day 8 onwards.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment with the test item.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects on hematology parameters were noted in females up to a 1000 mg/kg/day
No toxicologically relevant changes in hematology parameters were noted in males and females up to 1000 mg/kg/day.
Values in treated males and females achieving a level of statistical significance, when compared to concurrent controls, were considered to have arisen as a result of slightly low control values, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to have been affected by treatment up to 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in males and females up to 1000 mg/kg/day.
At 1000 mg/kg/day, males showed a minimal increase in sodium concentrations (1.01x of control).
Any differences in clinical chemistry parameters, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes in testosterone concentration in males and FSH, LH and estradiol concentrations in females were noted that were considered test item related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength and motor activity were similar between treated and control groups.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
For the thyroid gland in females treated at 1000 mg/kg bw/day an increased relative organ weight was noted, with no significant changes in absolute weight. There were no test item-related macroscopic of microscopic findings in the thyroid gland and the small increase was considered to be due to the small decrease in final body weight and not directly test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with Azodicarbonamide Unifoam AZ VI-50 were noted in the testes and epididymides of males.
In males, mild bilateral tubular degeneration/atrophy of the testes and mild bilateral cellular debris in the epididymides was present in two animals at 300 mg/kg/day and three animals at 1000 mg/kg/day. Moderate tubular degeneration/atrophy of the testis and mild cellular debris in the epididimydes was also observed in one vehicle-treated male.
One additional male treated at 1000 mg/kg bw/day showed marked bilateral spermatoid depletion in the testes and severely decreased sperm in the bilateral epididymides.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: marked spermatid depletion in the testes and severely decreased sperm in the epididymides of one male at 1000 mg/kg bw/day

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Any other information on results incl. tables

	Males
Dose level (mg/kg/day):	0	100	300	1000
Testes	10	10	10	10
Tubular degeneration/atrophy,    bilateral
Mild	-	-	2	3
Moderate	1	-	-	-
Spermatid depletion, bilateral
Marked	-	-	-	1
Epididymides	10	10	10	10
Cellular debris, bilateral
Minimal	-	-	2	3
Mild	1	-	-	-
Sperm decreased, bilateral
Severe	-	-	-	1

Applicant's summary and conclusion

Conclusions:

Based on the results of a 90-day study performed in rats according to OECD/EC guidelines and GLP principles, the sub-chronic NOAEL is concluded to be 300 mg/kg bw/day for males and at least 1000 mg/kg bw/day for females.

Executive summary:

A 90-day study was performed in rats according to OECD/EC guidelines and GLP principles, with oral exposure to ADCA at 0, 100, 300 or 1000 mg/kg bw/day. Accurate dosing was confirmed by analysis of the formulations. The following parameters and end points were evaluated in this study: clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrous stage, clinical pathology parameters (hematology, coagulation, clinical chemistry and hormone analysis), gross necropsy findings, organ weights, sperm analysis and histopathologic examinations.

At 100 mg/kg bw/day one female was found dead during the study period, however the cause of death was considered to be related to the gavage procedure. At 300 mg/kg bw/day, microscopic evaluation showed a test item-related but non-adverse minimal bilateral cellular debris in the epididymides (within historical range). At 1000 mg/kg bw/day, a test item-related but non-adverse minimal increase in sodium concentration in males was observed. Microscopic evaluation showed test item-related non-adverse mild bilateral tubular degeneration/atrophy of the testes and minimal bilateral cellular debris in the epididymides in three males (exceeding historical data range). Marked spermatid depletion in the testes and severely decreased sperm in the  epididymides of one additional male was observed. These findings were test item-related and considered adverse as it may have effects on the reproductive capacity of the animal. No test item-related microscopic alterations were observed in females.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical signs, functional observation, body weight, food consumption, ophthalmoscopy, hematology and coagulation parameters, hormone concentrations (testosterone, FSH, LH, estradiol), macroscopic examination and organ weights).

In conclusion, administration of Azodicarbonamide Unifoam AZ VI-50 by once daily oral gavage was well tolerated in Wistar Han female rats at levels of up to 1000 mg/kg bw/day. From the results presented in this report, the no-observed-adverse-effect level (NOAEL) was established to be 300 mg/kg bw/day for males and at least 1000 mg/kg bw/day for females.

There is no apparent correlation between the significant findings in the testis and epididymides of the single males of 1000 mg/kg/day and the findings in the males of 300 and 1000 mg/kg/day, and taking into account that there are no significant test-item related effects in the affected male in any of the other parameters assessed in the study, it cannot be concluded if the findings in single male of 1000 mg/kg/day are test-item related or not.