C,C'-azodi(formamide)

Endpoint summary

Description of key information

An oral 90-day study was performed in rats according to OECD/EC guidelines and GLP principles. Based on this study the sub-chronic NOAEL (oral route) is concluded to be 300 mg/kg bw/day for males and at least 1000 mg/kg bw/day for females. One dietary study on ADCA and on its main metabolite (biurea) is also available (Oser, 1965) and supports this result. 

Reliable sub-chronic inhalation studies in rats and mice are available. No effect has been observed and no target organ has been identified in both species after 13-week inhalation at the highest levels tested (200mg/m3). Therefore, the NOAEL in rats and mice after sub-chronic (13 weeks) inhalation is concluded to be 200mg/m3. 

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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).

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

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

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

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

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

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.

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.

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

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

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

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

	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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A reliable study is available (Klimisch 1).

System:

male reproductive system

Organ:

testes

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

lower number of animals, no evaluation of water consumption, no ophtalmological examination

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: F344/N

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 2week study: Frederick Cancer Research Facility (Frederick, MD); 13 week study: Simonsen laboratories, Inc. (Gilroy, CA)
- Age at study initiation: 7weeks (at the beginning of exposure)
- Weight at study initiation:no data
- Fasting period before study:no
- Housing:2/cage during acclimatization, 1/cage during main study.
- Diet (e.g. ad libitum): Zeigler NIH-07 Open Formula Rat Ration (Zeigler Brothers, Inc., Gardners, PA)
- Water (e.g. ad libitum): not mentioned but "automatic watering system" suppose ad libitum
- Acclimation period: 2 week study: 20-21 days, 13week study: 18-22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.2-25.4
- Humidity (%):70-85
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: 2-week: The overall, mean ADA concentration for each chamber was within 8% or less of target concentration. The relative standard deviation of daily means was within 16%. The aerosol had an average mass median aerodynamic diameter (MMAD) of 2.13 µm (range 1.89 to 2.45) with a mean geometric standard deviation of 1.9.

13-week: The overall mean ADA concentration for each chamber was within 3% or less of target concentration. The relative standard deviations of daily means were within 10%. The aerosol had an average MMAD of 2.38 µm (range 2.33 to 2.45), with a mean standard deviation of 1.7.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Stainless-steel, multitiered, whole-body exposure chambers (H1000, Hazelton Systems, Aberdeen, MD)
- Method of holding animals in test chamber: cages
- Source and rate of air:7±1 ft^3/min
- Method of conditioning air:
- System of generating particulates/aerosols:Jet-O-Mizer/screw feed method
- Temperature, humidity, pressure in air chamber:23.6°C, 70-85%, pressure not specified
- Air flow rate:7±1 ft^3/min
- Air change rate:12±2 air changes per hour
- Method of particle size determination:Lovelace multijet cascade impactor
- Treatment of exhaust air:not specified

TEST ATMOSPHERE
- Brief description of analytical method used:The aerosol concentration in the exposure chamber was monitored by sampling at a flow rate of 0.5 liter/min for three, 2-hr periods during the 6-hr exposure. Samples were collected on 25-mm fiberglass filters (Type AE, Gelman, Ann Arbor, MI). Each expsosure day, aerosol generation was started and sampling began after 12 min of rise time, when the chamber concentration had reached 90% of equilibrium concentration (T90). Therefore, the total exposure was 6 hr plus a T90 of 12 min. The control chamber was also sampled daily.

A RAM-S continuous aerolsol monitor (GCA, Bedford, MA) was used to monitor the stability of the aerosol concetration and to adjust the aerosol generator during exposure. It was operated on each chamber at the beginning, middle and end of the filter sampling period.
- Samples taken from breathing zone: not specified.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The aerosol concentration in the exposure chamber was monitored by sampling at a flow rate of 0.5 liter/min for three, 2-hr periods during the 6-hr exposure.
Samples were collected on 25-mm fiberglass filters (Type AE, Gelman, Ann Arbor, MI).
Each expsosure day, aerosol generation was started and sampling began after 12 min of rise time, when the chamber concentration had reached 90% of equilibrium concentration (T90).
Therefore, the total exposure was 6 hr plus a T90 of 12 min.
The control chamber was also sampled daily.

Duration of treatment / exposure:

2 weeks and 13 weeks

Frequency of treatment:

5d/w for 2 or 13 weeks

Dose / conc.:

2 mg/m³ air (nominal)

Remarks:

2-weeks exposure

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

2-weeks exposure

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

2-weeks and 13-weeks exposure

Dose / conc.:

100 mg/m³ air (nominal)

Remarks:

2-weeks and 13-weeks exposure

Dose / conc.:

200 mg/m³ air (nominal)

Remarks:

2-weeks and 13-weeks exposure

No. of animals per sex per dose:

2 week study: 5 animals
13 week study: 10 animals

Control animals:

yes

Details on study design:

- Dose selection rationale: 10 times higher than expected exposure to workers

Positive control:

no.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: morbidity/mortality checks

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: initiation, 1 week, and termination

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: termination
- Anaesthetic used for blood collection: Yes
- Parameters checked
For the 13-week study, blood was collected at necropsy from anesthetized rats and mice from the basic study group by cardiac puncture and placed in glass vials containing EDTA. A Coulter Electronics Model S-550 was used for analysis of erythrocyte count, mean corpuscular volume, haemoglobin concentration, hematocrit (calculated), and leukocyte count. Smears were made from the blood, stained with Wright’s stain, and examined under a light microscope to obtain differential leukocyte counts and counts of nucleated erythrocytes. Additional blood smears were stained with new methylene blue and examined for the presence of reticulocytes.

URINALYSIS: Yes
- Time schedule for collection of urine: 1 week prior to termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked:URINARY ENZYMES- In the 13-week study, 1 week prior to termination, all rats in the basic study group were placed in metabolism cages for overnight urine collection. Urine was collected on ice and analyzed for total amounts of lactate dehydrogenase (LDH), ß-galactosidase (ß-G), N-acetylglucosamididase (NAG), and alkaline phosphatise (AP). Lactate dehydrogenase was quantitated by using pyruvate as a substrate; 4-nitrophenylphophate was the substrate for alkaline phosphatise; p-nitrophenol was the substrate for both N-acetyl-ß-D-glucosaminidase and ß-galactosidase.

Sacrifice and pathology:

NECROPSY AND HISTOPATHOLOGY: Yes
All rats and mice in the basic study group, for both the 2-week and the 13-week studies, were given complete gross necropsy examinations. Rats and mice were killed by cardiac puncture exsanguination while under halothane anesthesia and were necropsied immediately. Weights of liver, thymus, right kidney, right testicle, brain, heart, and lungs (including trachea) were taken. Tissues were fixed in 10% neutral-buffered formalin. Tissues for microscopic examination were embedded in paraffin, sectioned at 5µm, and stained with hematoxylin and eosin.

The following tissues were trimmed and sectioned for histopathology: adrenals, bone (vertebra, with bone marrow and spinal cord; femur; rib), brain, epididymus or oviduct, esophagus, heart, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), both kidneys, larynx, liver, lung (4 lobes), lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary glands, nose (3 levels), pancrease (including islets), parathyroid gland, pituitary gland, prostate or uterus, salivary glands, seminal vesicles.

Other examinations:

SPERM MORPHOLOGY-VAGINAL CYTOLOGY. In the 13-week study, vaginal cytology samples were taken on a daily basis from basic study females for 1 week before final termination. Samples were obtained with sterile saline, placed on duplicate Dakin slides, fixed with Spray Cyte (Clay Adams 7180), and stained with toluidine blue (0.5% in 20% ethanol). Live sperm were obtained from the cauda of the right epididymis of male rats and mice at necropsy. Sperm were incubated at 37°C in Tyrode buffer, and viability was quantitated as the percentage of motile sperm in the sample. Sperm density (number of sperm per gram of caudal tissue) was quantified on a hemocytometer. The number of sperm in the sample was divided by the weight of the cauda of the epididymis to obtain the value for sperm density. Evaluations of sperm morphology were done using preparations of sperm fixed in ethanol and stained with eosin Y.

SPECIAL STUDY ENDPOINTS—Determinations of methemoglobin in whole blood of rats and mice exposed to ADA for 2 weeks were made using the method of Evelyn and Malloy (1938). Acetylcholinesterase concentrations in whole blood of rats only in the 13-week study and both rats and mice in the 2-week study were determined using the method of Ellman et al (1961), in which acetylthiocholine is used as the stubstrate. Serum thyroxine (T4 or tetraiodothyronine) and triiodothyronine (T3) levels for rats exposed to ADA for 13 weeks were measured by radioimmunoassay (Fietz, 1986).

Statistics:

All data were analyzed seperately for each sex.
Organ and terminal body weights on animals found dead or euthanized because of a moribund condition were not included in the statistical analysis.
Analysis of variance techniques were used for statistical evaluation.
Provided Bartlett's test of homogenetiy of variance was not significant, exposure groups were compared to controls by using Dunnett's multiple range test.
When Bartlett's test was significant, comparisons with the control group were made by a modified Student's t test, making allowance for unequal variance.
All statistical tests were conducted at a 5%, two-sided risk level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increase lung weight male 50 mg/m^3

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

2-week study
CLINICAL SIGNS AND MORTALITY
There were no abnormal clinical observations during the 2-week exposures for rats. No rats died.
BODY WEIGHT AND WEIGHT GAIN
The mean terminal body weights for the basic study male rats exposed to 200 mg/m3 ADA were significantly less than those of controls (95% of control value).

HAEMATOLOGY-CLINICAL CHEMISTRY
There were no significant differences in methemoglobin levels or acetylcholinesterase activities in whole blood of male and female rats exposed to any concentration of ADA, when compared to controls.

ORGAN WEIGHTS
The mean liver weights of male rats were significantly less at the 200 mg/m3 exposure than those of controls (83% of control values). Other organ weights of rats were not influenced by ADA exposure.
GROSS PATHOLOGY

HISTOPATHOLOGY: NON-NEOPLASTIC
A complete set of tissues from rats exposed to 200 and 0 mg ADA/m3 was examined histologically. The two exposure groups could not be distinguished on the basis of histological findings. Because no effect was seen at the highest exposure concentration, we did not examine tissues from rats exposed to lower concentrations.


13- Week Study
CLINICAL SIGNS AND MORTALITY
There were no abnormal clinical observations during the 13-week exposures for rats that could be attributed to exposure to ADA. No deaths occurred that could be attributed to ADA. One female rat (50 mg/m3 exposure group) was euthanized because of weight loss and dehydration.

BODY WEIGHT AND WEIGHT GAIN
The mean terminal body weights for the basic study rats exposed to ADA were not significantly different from those of the controls.

HAEMATOLOGY
Results obtained for hematology in rats exposed to ADA for 13 weeks indicated that there were no exposure-related alterations in blood parameters. There were no significant, exposure-related changes, in either male or female rats, in the amounts of the four urinary enzymes (LDH, AP, beta-G, NAG) excreted.

ORGAN WEIGHTS
Lung weights of male and female rats were increased (111% of control) at the 50 mg/m3 exposure level.

HISTOPATHOLOGY: NON-NEOPLASTIC
A complete set of tissues from rats exposed to 200 and 0 mg ADA/m3 was examined histologically. Although several incidental lesions were present in both groups, lesions attributable to the ADA exposure were not present.
Five male and six female rats exposed to 50 mg/m3 ADA were described as having enlarged bronchial or bronchial and mediastinal lymph nodes at gross necropsy. Histological examination of these nodes showed a moderate-to-severe lymphoid hyperplasia. Subsequently, the lungs of all rats and mice in the 50 mg/m3 exposure group were examined histologically. All male and female rats in the 50 mg/m3 exposure group euthanized at the end of the 13-week exposure period had a spectrum of lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type II cell hyperplasia that was associated with a moderate number of mixed inflammatory cells.
The extent of lung involvement varied from mild to moderate among animals. In some animals, the perivascular cuffs were predominant, while other animals showed both the perivascular cuffs and the epithelial hyperplasia. The lungs from all rats and mice exposed to 100 mg ADA/m3, as well as the lungs of mice exposed to 50 mg/m3, were examined.
There were no significant lesions on histologic examination.

Samples of lung and kidney of male rats exposed to ADA for 13 weeks were analyzed for the presence of ADA and biurea. These tissues were chosen, because they are the most likely tissues to contain significant quantities of one or both compounds, lung being the organ of exposure and kidney, because biurea had been detected in kidney.
No ADA was detected in either lung or kidney of male rats exposed to ADA for 13 weeks. Biurea, however, was detected in lungs, but not kidney, of rats exposed to 50, 100, and 200 mg/m3 ADA. The amount of biurea in the lungs increased nonlinearly with increasing exposure concentration. The percentage of biurea retained in lungs was calculated as a percentage of ADA deposited on the last day of exposure. Although 66% of the amount of ADA expected to be deposited in rats exposed to 200 mg/m3 on the last exposure day is retained in the lungs as biurea, this is a small percentage (1%) of the total amount of ADA deposited over the entire study.

OTHER FINDINGS
There were no adverse effects from inhalation exposure to ADA for 13 weeks, with respect to right caudal weight, right epididymal weight, right testicular weight, sperm motility, sperm count per gram caudal tissue, or incidence of abnormal sperm. However, there was a small, but significant, increase in sperm count in male rats exposed to 50 or 100 mg ADA/m3.

There were no apparent adverse effects on menstrual cyclicity or on estrous cycle length in any of the dose groups, except in 2 out of the 10 animals in the 200 mg/m3 dose group. For these animals, estrous cycle length was >7 days or was not precisely determined.

There were no significant differences in acetylcholinesterase activities in whole blood of male and female rats exposed to any concentration of ADA when compared to controls. T3 and T4 levels in serum from male rats increased with increasing ADA exposure concentration in male rats. T3 and T4 levels in the highest exposure group were significantly elevated, relative to control. T3 was increased approximately 50%,while T4 was increased approximately 40%. T3 and T4 were unchanged in female rats exposed to ADA.

Dose descriptor:

NOAEC

Effect level:

200 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose level tested

Critical effects observed:

no

Table 1: 13 -weeks microscopic observations in lungs and lymph nodes of rats.

		Males	Males	Males	Males	Females	Females	Females	Females
	Exposure group (mg/m^3)	0	50	100	200	0	50	100	200
	No. in Group	10	10	10	10	10	9	10	10
	Microscopic observations
Lungs		10	10	10	10	10	9	10	10
	inflammation; nonsuppurative; alveolus	0	10	0	0	0	8	0	0
	Hyperplasia; alveolar epithelium	0	10	0	0	0	8	1	0
	Infiltrating cell; lymphocytic plasmacytic; perivascular	0	10	0	1	0	9	1	0
	Hemorrhage; multifocal, acute; alveolus, right apical	0	0	0	1	0	0	0	0
Tracheobronchial lymph nodes (number examined)		8	10	0	7	9	9	0	7
	Hemorrhage; acute; sinus	0	0	0	0	1	0	0	0
	Hyperplasia; lymphoid	0	7	0	0	0	7	0	0
Mandibular lymph nodes (number examied)		9	0	0	9	9	0	0	10
	Hemorrhage; acute; sinus	0	0	0	0	1	0	0	0
	Hyperplasia; lymphoid; follicular	0	0	0	0	1	0	0	0
Mediastinal LN(number examined)		7	10	0	7	7	9	0	7
	Hemorrhage; acute; sinus	2	0	0	1	2	0	0	6
	Hyperplasia; lymphoid	0	5	0	0	0	4	0	0

Table 2: air concentrations

SUMMARY OF AIR CONCENTRATIONS AND PARTICLE SIZES IN EXPOSURE CHAMBERS FOR RATS AND MICE EXPOSED TO AZODlCARBONAMIDE
	2-week repeated			13-week subchronic
Target exposure concentration	Rats	Mice	Particle size	Rats	Mice	Particle size
mg/m3	mg/m3	mg/m3	MMAD SD	mg/m3	mg/m3	MMAD SD
Control	0	0	-	0	0	-
2	2.0 (13)	2.1 (12)	1.89 2.1	-	-	-
10	9.4 (11)	9.6 (10)	1.95 1.8	-	-	-
50	52 (10)	52 (7)	2.15 1.8	50(10)	50 (10)	2.33 1.8
100	102 (16)	102 (15)	2.22 1.7	100(7)	100 (7)	2.45 1.7
200	207 (10)	217 (11)	2.43 1.9	204(5)	204 (5)	2.37 1.8

Table 3:

LEVELS OF ADA AND BlUREA IN LUNGS AND KIDNEYS OF MALE RATS EXPOSED TO AZODICARBONAMIDE (ADA) FOR 13 WEEKS
Exposure level(mg/m3)	ADA		Biurea
	Kidney	Lungs andbronchi	Kidney	Lungs andbronchi
0	NP b	NP	NP	NP
50	ND e	ND	ND	79± 15 d
100	ND	ND	ND	303± 79
200	NP	NP	NP	948 ±238
b NP, no peak was observed at the expected retention time. Limit of quantitation is 100µg per sample.
e ND, not determined.
d Mean µg/g tissue ± standard deviation; n = 5

Conclusions:

In summary, ADA is rapidly cleared from the lungs, even when inhaled at concentrations up to 200 mg/m^3. Exposure to ADA for up ot 13 weeks did not appear to be toxic to rodents.

Executive summary:

Two-week repeated and 13-week sub-chronic inhalation exposures of F344/N rats to Azodicarbonamide (named ADA) were conducted to determine the toxicity after exposure via inhalation. No exposure-related mortality or abnormal clinical signs were observed in rats during or after exposure. The terminal body weights were slightly depressed in the highest exposure group. Liver weights were lower in male rats exposed to 200 mg ADA/m3. No significant lesions were noted on either gross or histologic evaluation of rats or mice. In the 13-week sub-chronic study, the mean air concentrations of ADA were 204, 100, or 50 mg/m3. No mortality or clinical signs related to exposure were observed. The terminal body weights of exposed rats were not significantly different from those of control rats but were significantly depressed in mice exposed to 100 or 200 mg ADA/m3. No histopathological lesions were noted. Lung weights were increased and enlarged mediastinal and/or tracheobronchial lymph nodes were noted in rats exposed to 50 mg ADA/m3. No exposure-related lesions were observed microscopically in rats exposed to 100 or 200 mg ADA/m3. All rats in the 50 mg ADA/m3 exposure group only had lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type II cell hyperplasia, suggesting a possible immune reaction to an antigen in the lung. The possibility of an unknown viral antigen causing this lesion cannot be excluded. Lung tissue from male rats was analyzed for ADA and biurea, the major metabolite of ADA. No ADA was detected. The amount of biurea in the lungs increased nonlinearly with increasing exposure concentration, suggesting that clearance was somewhat impaired with repeated exposures. However, even at the highest exposure concentration, this amount of biurea was less than 1% of the estimated total deposited over the exposure period. In summary, ADA is rapidly cleared from the lungs, even when inhaled at concentrations 1% to 200 mg/m3. Exposure for up to 13 weeks did not appear to be toxic to rodents. The NOAEC was 200mg/m3 in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

200 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available study is reliable (Klimisch 2).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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.

These results are supported by those of Oser studies on ADCA and metabolites (1965).

In the Medinsky study (1990), 2 -week and 13-week repeated sub-chronic inhalation exposures of F344/N rats and B6C3F1 mice to Azodicarbonamide (ADA) were conducted to determine the toxicity of inhaled ADA. The mean air concentrations of ADA in the 2-week studies were 207, 102, 52, 9.4, or 2.0 mg/m3. No exposure-related mortality or abnormal clinical signs were observed in rats or mice during or after exposure. The terminal body weights were slightly depressed in the highest exposure group. Liver weights were lower in male rats exposed to 200 mg ADA/m3. No significant lesions were noted on either gross or histologic evaluation of rats or mice. In the 13week sub-chronic study, the mean air concentrations of ADA were 204, 100, or 50 mg/m3. No mortality or clinical signs related to exposure were observed. The terminal body weights of exposed rats were not significantly different from those of control rats but were significantly depressed in mice exposed to 100 or 200 mg ADA/m3. No histopathological lesions were noted in mice. Lung weights were increased and enlarged mediastinal and/or tracheobronchial lymph nodes were noted in rats exposed to 50 mg ADA/m3. No exposure-related lesions were observed microscopically in rats exposed to 100 or 200 mg ADA/m3. All rats in the 50 mg ADA/m3 exposure group only had lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type II cell hyperplasia, suggesting a possible immune reaction to an antigen in the lung. Lung tissue from male rats was analyzed for ADA and biurea, the major metabolite of ADA. No ADA was detected. The amount of biurea in the lungs increased nonlinearly with increasing exposure concentration, suggesting that clearance was somewhat impaired with repeated exposures. However, even at the highest exposure concentration, this amount of biurea was less than 1% of the estimated total ADA deposited over the exposure period. In summary, ADA is rapidly cleared from the lungs, even when inhaled at concentrations up to 200 mg/m3. Exposure to ADA for up to 13 weeks did not appear to be toxic to rodents.

 

Justification for classification or non-classification

Based on the current data-set, ADCA is not classified for STOT-RE according to Regulation (EC) No 1272/2008.