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REACH

C,C'-azodi(formamide)

EC number: 204-650-8 | CAS number: 123-77-3

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A one-generation study and a three-generation study performed with the registered substance are available. The results of these studies do not indicate reproductive toxicity.

In a repeated dose 90-day oral toxicity study (OECD 408) at 1000 mg/kg bw/day, microscopic evaluation showed test item-related 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 can be expected to affect the reproductive capacity of the animal. Thus a study performed to OECD 443 (ECHA basic design; 10-week pre-mating period, without extension of the Cohort 1B; without Cohorts 2 and 3 ) is proposed.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:: experimental study planned
Study period:: The study will be performed subject to confirmation of the the testing proposal by ECHA.
Justification for type of information:: TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: azodicarbonamide (ADCA), EC 204-650-8, CAS 123-77-3

- Name of the substance for which the testing proposal will be used: azodicarbonamide (ADCA), EC 204-650-8, CAS 123-77-3

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- Available GLP studies: no GLP studies are available that are adequate to meet the information requirement (toxicity to reproduction) for this tonnage band (Annex X). A GLP one-generation reproductive toxicity study (OECD 415) is available and does not indicate any concerns in relation to the reproductive toxicity of the substance, but is not sufficient to meet the information requirement at this tonnage band.

- Available non-GLP studies: no non-GLP studies are available that are adequate to meet the information requirement (toxicity to reproduction) for this tonnage band (Annex X). A non-GLP 3-generation study performed with ADCA-treated flour is available and does not indicate any concerns in relation to the reproductive toxicity of the substance, but is not sufficient to meet the information requirement at this tonnage band.

- Historical human/control data: no relevant human data are available.

- (Q)SAR: no validated methods are available. (Q)SAR methods are not sufficiently robust to meet
the information requirement (toxicity to reproduction) for this tonnage band (Annex X)

- In vitro methods: no validated in vitro methods are available. The available in vitro methods are not sufficiently robust to meet the information requirement (toxicity to reproduction) for this tonnage band (Annex X)

- Weight of evidence: Insufficient data (i.e. GLP studies, non-GLP studies, human data, (Q)SAR methods, in vitro methods) are available to enable this information requirement to be met.

- Grouping and read-across: No suitable read-across substance(s) with adequate reproductive toxicity data have been identified. Azodicarbonamide (ADCA) is readily converted to biurea (1,2-hydrazinedicarboxamide, CAS 110-21-4), the only breakdown product identified. Studies in whole blood showed the total conversion of ADCA to biurea within five minutes. Consequently, systemic exposure will be to the metabolite and not to ADCA. However, adequate reproductive toxicity data are not available for the metabolite, and so read-across is not possible on this basis.

- Substance-tailored exposure driven testing: Not applicable. The supported uses of the substance are such that substance-tailored exposure-driven testing as defined in Annex XI are not applicable.

- Approaches in addition to above: Not applicable

- Other reasons: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- The substance is not known to be a genotoxic carcinogen. The substance is not known to be a germ cell mutagen. The substance is not of low toxicological activity, with no (or no significant) human exposure as defined in Column 2 of Annex X. Specific adaptation of the information requirement is therefore not possible.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- A study performed to OECD 443 (ECHA basic design; 10-week pre-mating period, without extension of the Cohort 1B; without Cohorts 2 and 3 ) is proposed.
Qualifier:: according to guideline
Guideline:: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:: The study will be performed to the current version of the test guideline, and following the ECHA 'basic' test design
Justification for study design:: SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals:
A standard pre-mating exposure period of 10 weeks will be used for both sexes. The 10-week premating dosing period is intended to cover full periods of spermatogenesis and folliculogenesis before mating, thereby allowing a meaningful assessment of the effects on fertility, and also allowing a conclusion on classification and labelling (including categorisation) for the hazard endpoint.

- Basis for dose level selection:
The dose levels for the proposed EOGRTS will be selected taking into account the available repeated dose toxicity data in the rat, including the existing 90-day oral repeated dose and one-generation reproductive toxicity studies, and also taking into account the regulatory limit dose of 1000 mg/kg bw/d. Dose levels selection will allow a conclusion on classification and labelling (including categorisation) for the CLP hazard endpoint (sexual function and fertility).

- Inclusion/exclusion of extension of Cohort 1B
The extension of Cohort 1B to include the F2 generation is required under the following conditions:

a) The substance has uses leading to significant exposure of consumers or professionals, taking into account consumer exposure from articles, and;

b) any of the following conditions are met:

The substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or;
There are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, or;
There are indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches.

While the uses of the substance may result in significant professional and/or consumer exposure (condition (a)), the substance does not meet any of the hazard triggers (condition (b)).
Although the substance shows genotoxic activity in vitro, somatic cell mutagenicity tests in vivo (bone marrow micronucleus and comet assays) are available and report negative results. The data demonstrate that the substance does not cause gene mutations or chromosomal aberrations; and the substance is not classified (or proposed to be classified) as Mutagen Category 2.

There is no indication from the available dataset that the substance and/or its metabolite(s) would reach steady state only after an extended exposure. The Log Kow is demonstrated experimentally to be <1.0. Bioaccumulation is therefore unlikely for the substance. Furthermore, toxicokinetic data for the substance clearly demonstrates the very rapid and complete metabolism of the substance and excretion of its metabolites. There is no indication from the existing in vivo studies that, after longer exposure duration, the effects of the substance are more severe or occur at lower dose than would be expected based on assessment factors generally used to extrapolate between studies of different exposure duration.

There are no data specifically investigating endocrine-disrupting (ED) properties of the substance;
however, relevant endpoints assessed in the available mammalian toxicity studies do not indicate
an ED mode of action (MoA). Findings in the 90-day repeated dose rat study (OECD 408) were seen in the testes; however a relationship to treatment was not conclusive. Furthermore, there were no effects on organ weight or histopathology of other endocrine-sensitive organs or tissues at any dose level investigated. A one-generation reproductive toxicity study (OECD 415) did not identify any effects on fertility or reproduction at the highest dose level tested (1000 mg/kg bw/d). There are no data from structurally similar substances which raise concerns in relation to an ED MoA. Taking into account the hazard profile of the substance, extension of Cohort 1B to include the F2 generation is not required according to ECHA REACH Guidance, and is not proposed.

- Termination time for F2
Not relevant, as extension of Cohort 1B to include the F2 generation is not required.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B
Triggers for the inclusion of Cohort 2 (developmental neurotoxicity, DNT) are based on a particular concern for (developmental) neurotoxicity. A particular concern may result from existing information on the substance; specific mechanisms/modes of action (MoA) associated with DNT; or similar in formation from structural analogues.

The acute oral toxicity study for the substance in the rat does not report specific clinical signs.
The only signs reported in this study (at the dose level of 5000 mg/kg bw) were transient and non-specific and do not indicate neurotoxicity. Similarly, no clinical signs indicative of neurotoxicity were observed in the acute dermal and acute inhalation toxicity studies. No clinical signs indicative of neurotoxicity and no evidence of neurohistopathology was reported in the 90-day and one-generation studies. In conclusion, there is not sufficient evidence, weighing all the information, to raise a reasonable expectation that the substance could be a developmental neurotoxicant. Inclusion of the developmental neurotoxicity cohorts (Cohort 2) in the study is not proposed.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3
Triggers for the inclusion of Cohort 3 (developmental immunotoxicity, DIT) are based on a particular concern for (developmental) immunotoxicity. A particular concern may result from existing information on the substance; specific mechanisms/modes of action (MoA) associated with DIT; or similar information from structural analogues. The available toxicity studies do not indicate a potential for immunotoxicity. The substance is a respiratory sensitiser, an MoA that involves the immune system, but this specific MoA does not raise any concerns of relevance to DIT. There is no evidence that the substance or its structural analogues has any potential to cause developmental immunotoxicity. In conclusion, there is not sufficient evidence, weighing all the information, to raise a reasonable expectation that the substance could be a developmental immunotoxicant. Inclusion of the developmental immunotoxicity cohorts (Cohort 3) in the study is not proposed.

- Route of administration
The oral route will be used, as the standard route envisaged by ECHA REACH Guidance and taking into account the physicochemical properties of the substance.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals
The study will be performed in the rat as the standard species identified by the OECD test guideline and by ECHA REACH Guidance. The strain of rat will be a standard strain used in regulatory toxicity studies, to be guided by the available toxicity data for the substance and the experience of the CRO. The dosing vehicle will be from a standard list and will be selected based on solubility of the substance and the experience of the CRO. The number of animals per group will be selected on the basis of the experience of the CRO and to meet the requirement of the test guideline, that each test and control group should contain a sufficient number of mating pairs to yield at least 20 pregnant females per dose group.

Reference 2

Endpoint:: multi-generation reproductive toxicity
Type of information:: experimental study
Adequacy of study:: weight of evidence
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Study well documented, although some data are missing (purity, for example)
Reason / purpose for cross-reference:: reference to same study
Qualifier:: no guideline followed
Principles of method if other than guideline:: A multigenerational study has been realised on animals treated in diet for 2 years. Animals were also evaluated for carcinogenicity and chronic effects.
GLP compliance:: no
Limit test:: no
Species:: rat
Strain:: other: FDRL albino
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Food Research Laboratories' stock colony originated in 1928 with breeders of the Wistar strain
- Age at study initiation: 28 days old
- Weight at study initiation: 50 and 70 g
- Fasting period before study: no
- Housing: individually in suspended wire-mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: bred in the laboratory

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-26°C (76±2°F)
- Humidity (%):50±10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Route of administration:: oral: feed
Vehicle:: other: bread made with untreated flour
Details on exposure:: Basal Diet composition:
Bread (air-dried) 83%, Casein 10%, Salt mixture 3%, Primex 2%, Vitamin mixtures 2%

Bread was made of the following formula: flour 50,non fat milk solids 3, sugar 2, yeast 1, shortening 1, salt 1, malt, 0.375, yeast food 0.5, monoglycerides 0.75.
Primex from Procter and Gamble Distributing Company, New York.
Details on mating procedure:: - M/F ratio per cage:1/2
- Length of cohabitation: 21days
- Proof of pregnancy: recognized visually, by palpation, or from weight increments
- After 21 days of unsuccessful pairing replacement of first male by another male with proven fertility. If pregnancy was not established by the third week, the male was replaced. Following three (or occasionally 4) unproductive trials with females of known fertility, males were considered sterile and retired. Females were continued for a minimum of 6 matings with fertile males, even though some failures may have intervened.
- After successful mating each pregnant female was caged (how): the females were transferred to individual cages.
Analytical verification of doses or concentrations:: not specified
Duration of treatment / exposure:: 2 years
Frequency of treatment:: in diet
Details on study schedule:: The reproduction and lactation program was initiated shortly after the 12th week on the test diets when the rats were approximately 110 days old and, of course, sexually mature.

After weaning of the second litter of the F0 rats, matings were discontinued and the rats remained on test for the 2-year period.
From the young of the second (F1b) litters ten male and ten female pups in each treatment group were selected to continue on their respective diets for observations similar to those described above for the initial 12-week period. Subsequently, they were mated and carried through weaning of their second litters and the cycle was repeated through an F2 generation and again through the initial 12-week growth phase of the F3 generation.
This approximated the end of the 2-year period for-the F0 rats, when all rats were sacrificed. At this time the F1 and F2 rats completed 28 or more weeks, and F3 rats 14 weeks, on test diets.

Lactation was permitted for three weeks. Following weaning, death, or destruction of their litters, the females were allowed a one-week rest period before remating. In successive matings, the males were rotated among the females within their respective test groups.

Matings continued in the F0 generation throughout the entire two-year period. First litters were discarded at weaning. From the second litters of as many different mothers as possible, 10 rats of each-sex were selected whose individual weights approximated the averages for their respective litters. These F1 generation animals were raised to maturity and mated like the parent generation. The second litters of the F2 generation were carried through the same breeding program. Similarly, representative rats of the F3 generation were raised to maturity for growth observations but not mated because the entire study was terminated when the F0 rats reached two years on test.
Remarks:: Doses / Concentrations:
0, 7.5ppm
Basis:
nominal in diet
ADCA
Remarks:: Doses / Concentrations:
0, 750, 3750, 7500ppm
Basis:
nominal in diet
Buirea
No. of animals per sex per dose:: six experimental groups 25 males and 25 females each
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: 7.5ppm is 10 times higher than the intended use level of ADCA.
Positive control:: None
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: During the reproduction phase of the experiment body weights were recorded biweekly; females were also weighed at the time of mating and at appropriate intervals to confirm pregnancy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes, but no furter data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 6, week 12, and subsequently at semiannual periods
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: in representative animals
- Parameters examined: hemoglobin, hematocrit, total and differential leukocytes counts, glucose and non-protein nitrgen levels.

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: week 6, week 12, and subsequently at semiannual periods
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: albumin, sugar, and pH, and the sediment was examined microscopically

NEUROBEHAVIOURAL EXAMINATION: No
Oestrous cyclicity (parental animals):: no
Sperm parameters (parental animals):: no
Litter observations:: Pups were weighed at 4, 12 and 21 days of age at which time they were weaned.
Postmortem examinations (parental animals):: Necropsies were performed on all rats.

GROSS PATHOLOGY: Yes
Gross abnormalities were noted and weights were recorded for liver, kidneys, spleen, heart, and adrenal glands.

HISTOPATHOLOGY: Yes
Histopathologic examinations were made of the liver, kidneys, spleen, pancreas, adrenals, lungs, heart, gonads, stomach, large and small intestines, bladder, lymph nodes, salivary, thyroid, and pituitary glands, and all visible abnormalities.
Postmortem examinations (offspring):: Growth and development of offspring were evaluated.
Reproductive indices:: - fertility index (F.I.), the percentage of matings resulting in pregnancy; Fertility index = (pregnancies/matings) x100
- gestation index (G.I.), the percentage of pregnancies resulting in the birth of live litters; Gestation index = (litters born/pregnancies) x 100
Offspring viability indices:: - viability index (V.I.), the percentage of rats born that survived 4 days or longer; Viability index = (pups surviving at 4 days/pups born) x 100
- lactation index (L.I.), the percentage of rats alive at 4 days that survived the 21-day lactation period. Lactation index = (pups weaned/pups at 4 days) x 100
Clinical signs:: no effects observed
Description (incidence and severity):: see chronic toxicity section
Body weight and weight changes:: no effects observed
Description (incidence and severity):: see chronic toxicity section
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: see chronic toxicity section
Organ weight findings including organ / body weight ratios:: no effects observed
Histopathological findings: non-neoplastic:: no effects observed
Description (incidence and severity):: see chronic toxicity section
Other effects:: not specified
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: not examined
Reproductive performance:: no effects observed
Dose descriptor:: NOAEC
Remarks:: Reproduction
Effect level:: 7 500 ppm (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: No effects observed up to and including the highest dose level tested (7500 ppm)
Critical effects observed:: no
Clinical signs:: no effects observed
Description (incidence and severity):: see chronic toxicity section
Mortality / viability:: no mortality observed
Description (incidence and severity):: see chronic toxicity section
Body weight and weight changes:: no effects observed
Description (incidence and severity):: see chronic toxicity section
Sexual maturation:: no effects observed
Description (incidence and severity):: see chronic toxicity section
Organ weight findings including organ / body weight ratios:: no effects observed
Description (incidence and severity):: see chronic toxicity section
Gross pathological findings:: no effects observed
Histopathological findings:: no effects observed
Dose descriptor:: NOAEC
Generation:: F1
Effect level:: 7 500 ppm (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: No effects observed up to and including the highest dose level tested (7500 ppm)
Critical effects observed:: no
Reproductive effects observed:: no

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc., Tsukuba Rearing Center
- Age at study initiation: Males at 3 weeks, Females at 6 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: Each animal was individually housed in a metallic cage with a metallic mesh floor (220w x 270d x 190h mm) in an animal room (Room 15).
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Animals were given free access to pellet food (CE-2, Japan Clea Inc.)
- Water (e.g. ad libitum): Animals were given free access to drinking water (tap water supplied by Hatano Waterworks Department).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24+/-1ºC
- Humidity (%):50-65%
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Nakalai Tesque, Inc.; V8N6540 and V9F1299

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The stability of dosing suspensions was evaluated by comparing the absorption spectrum obtained at 300-600 nm to that obtained with ADC suspensions prepared at the same concentrations as dosing suspensions. The results obtained showed that 20 and 200 mg/mL dosing suspensions were stabile for at least 8 days in refrigerator when protected from light. It was therefore decided to prepare dosing suspensions at least once a week and to store them in refrigerator protected from light and consume them within 7 days after preparation. In addition, the homogeneity and content of ADC were evaluated once (homogeneity) or twice (content) during the study period by determining absorption at 424 nm. The results obtained indicated that thoroughly stirred dosing suspensions evenly contained the designated amount of ADC.

Daily treatment was conducted in the designated time zone (between 9:00 and 13:00). The dosing volume (5 mL/kg) for each animal was calculated based on body weight determined once a week in males. In females, the dosing volume was calculated based on body weight determined once a week before and during the mating period, on days 0, 7, 14 and 20 of gestation (day 0 of gestation = day on which vaginal plugs or sperms were detected) after copulation was confirmed, and on days 0, 4, 7 and 14 of lactation after parturition.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle, corn oil was added to ADC, weighed to attain each concentration, and the mixture was stirred to suspend.
- Concentration in vehicle: The ADC content was adjusted so that the dosing volume was 5 mL/kg of body weight regardless of the dose.
- Purity: 99.5 wt%

Details on mating procedure:

Animals were stratified based on body weight determined the day before starting administration (males) or the day before starting observation of the estrous cycle (females) and randomly assigned to groups of 25 males and 25 females.
- M/F ratio per cage: 1:1
- Length of cohabitation: 3 weeks
- Proof of pregnancy: vaginal plug referred to as day 0 of gestation
- Further matings after two unsuccessful attempts: not required
- After successful mating, females were separated from males and housed individually starting from the day copulation was confirmed (gestation day 0).

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

In males, 98 consecutive days until the day before necropsy: maximum of 3 week-mating period starting 10 weeks before starting mating.
In females, 2 weeks before starting mating, during the mating period (until copulation was confirmed), during the gestation period, and during a 20-day lactation period (day 0 of lactation=day of parturition).

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

The doses used in the present study were selected based on the results of an oral one-generation reproductive toxicity study of ADC in rats (preliminary dose-finding study; protocol No.: R-98-012).
Briefly, ADC, purchased from Tokyo Chemical Industry Co., Ltd. (lot No. GF01) and suspended in corn oil, was administered daily to groups of 5 males and 5 females of the same strain as that used in the present study at 0, 250, 500 or 1,000 mg/kg for 2 weeks before mating.
Females were necropsied on day 14 of gestation to determine the numbers of corpus lutea and implantation scars and the embryonic viability.
Males were necropsied the day after day 28 of treatment, and the testes and prostate ventral lobe were weighed.

No male or female showed any toxic signs or symptoms and their reproductive performance was not affected regardless of the dose.
The oral LD50 of ADC in rats was high (6400 mg/kg), indicating that ADC shows no marked toxic effects in rats.
Based on these findings, the highest dose was set at 1,000 mg/kg, followed by 300 mg/kg (intermediate dose) and 100 mg/kg (low dose) with a common ratio of about 3.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the treatment period, animals were observed before and after administration
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS:No data

BODY WEIGHT: Yes
- Time schedule for examinations: All males were weighed once a week on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 92 of treatment and the day of necropsy.
All females were weighed once a week until copulation was confirmed (days 1, 8, 15 and 22 of treatment). After copulation was confirmed, they were weighed on days 0, 7, 14 and 20 of gestation and on days 0, 4, 7, 14 and 21 of lactation following parturition. Body weight data obtained on day 22 of treatment were excluded from evaluation since they were obtained only from animals in which copulation had not been confirmed.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: In males, on days 2-3, 9-10, 16-17, 23-24, 30-31, 37-38, 44-45, 51-52, 58-59, 65-66, 71-72, 79-80 and 86-87 of treatment. However, data obtained on days 79-80 and days 86-87 were excluded from evaluation since animals were in the mating period and feeding condition differed from one animal to another. In females, on days 2-3 and 9-10 before mating, days 0-7, 7-14 and 14-20 of gestation after confirmation of copulation and days 0-4, 4-7, 7-14 and 14-21 of lactation after parturition.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):

Vaginal smear specimens were prepared and examined in all females from 2 weeks before starting treatment through the mating period until the day copulation was confirmed.
The estrous cycle was divided into three staged, estrus, early estrus and diestrus, based on the cellular composition of vaginal smears.
Females showing estrus only once during each of the two 2-week periods, before and after starting treatment, were regarded as monestrous.
In each of the other animals, the number of days between one estrus (the final day, if estrus was constant) and the next estrus was counted to calculate the mean number of days needed for the return of estrus.
The estrous cycle was classified into the “4-day cycle” if estrus returned in 4 days, the “5-day cycle” if estrus returned in 5 days, and the “4/5-day cycle” if both 4-day and 5-day intervals were found.
The estrous cycle was classified as irregular in all other cases.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.The size was not adjusted if it was less than 8.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: Dead newborns were subjected to necropsy after determining the presence or absence of external abnormalities and fixed in 10% formalin solution for storage. The lungs were excised from newborns found dead on day 0 after birth and placed in saline. If the lungs floated in the saline, the newborn was considered as stillborn. Dead newborns with major injury or maceration were fixed for storage without any further processing.

Postmortem examinations (parental animals):

GROSS NECROPSY
- Gross necropsy: external and internal examinations including the cervical, thoracic, and abdominal viscera.
In dead males, major thoracic and abdominal organs were macroscopically examined to determine the presence or absence of abnormalities, and major thoracic and abdominal organs were excised including the pituitary glands, thyroids, testes, epididymides, coagulating glands, seminal vesicle, and prostate.

In females which died or were sacrificed in extremis, major thoracic and abdominal organs were macroscopically examined to determine the presence or absence of abnormalities, and major thoracic and abdominal organs including the pituitary glands, thyroids, ovaries, uterus, cervix and vagina were excised.

HISTOPATHOLOGY / ORGAN WEIGHTS
MALES: The excised testes and epididymides were fixed in Bouin's solution (0.1M phosphate-buffered 10% formalin solution for long-term storage). All other excised organs and tissues were fixed in 0.1M phosphate-buffered 10% formalin solution for storage.
Organs and tissues with abnormalities, as well as the pituitary glands, thyroids, testes, epididymides, coagulating glands, seminal vesicle and prostate ventral lobe, were processed according to the conventional method to prepare paraffin sections, which were then stained with hematoxylin-eosin to prepare specimens for histopathological examinations.
These sections were prepared and examined for all animals.

All animals which serviced until day 98 of treatment were exsanguinated to death the following day under anesthesia with sodium pentobarbital and subjected to necropsy (routine anatomy). During necropsy, major thoracic and abdominal organs were macroscopically examined to determine the presence or absence of abnormalities. Tissues with abnormalities were fixed in 0.1M phosphate-buffered 10% formalin solution for storage. The pituitary glands, thyroids, testes, epididymides, coagulating glands, seminal vesicle and prostate, as well as the kidneys, stomach, spleen, thymus and adrenals, which showed abnormalities in females sacrificed in extremis before males were necropsied, were macroscopically examined to determine the presence or absence of abnormalities. The testes and epididymides were fixed in Bouin’s solution (0.1M phosphate-buffered 10% formalin solution for long-term storage). All other organs and tissues were fixed in 0.1M phosphate-buffered 10% formalin solution for storage. Tissues with abnormalities were processed to prepare paraffin sections according to the conventional method and stained with hematoxylin-eosin to prepare specimens for histopathological examinations. Specimens for histopathological examinations were prepared for the pituitary glands, thyroids, testes, epididymides, seminal vesicle, prostate ventral lobe and coagulating glands from all animals. All males from the control and high-dose groups, as well as males whose mates copulated but did not become pregnant in the mid and low-dose groups, were examined. Specimens for histopathological examinations were also prepared for the kidneys, which showed enlargement in necropsy in the high-dose group, in all males from the control and high-dose groups.

FEMALES: Excised ovaries were fixed in Bouin's solution (0.1M phosphate-buffered 10% formalin solution for long-term storage), while the uterus was fixed in 0.1M phosphate-buffered 10% formalin solution together with other tissues after counting the number of implantation scars. Organs and tissues with abnormalities, as well as the pituitary glands, thyroids, ovaries, uterus, cervix and vagina, were processed according to the conventional method to prepare paraffin sections and stained with hematoxylin-eosin to prepare specimens for histopathological examinations of all females.

Surviving animals were exsanguinated to death under anesthesia with an overdose of sodium pentobarbital and necropsied, those which showed parturition on day 21 of lactation and those which copulated but did not show parturition on presumed day 25 of gestation. Major thoracic and abdominal organs were macroscopically examined to determine the presence of absence of abnormalities. Tissues with abnormalities were fixed in 10% phosphate-buffered formalin solution for storage. The uterus was excised and fixed in 0.1M phosphate-buffered 10% formalin solution for storage after macroscopically counting the number of implantation scars. In addition to these organs, the pituitary glands, thyroids, ovaries, uterus, cervix and vagina, as well as the kidneys, heart, stomach, spleen, thymus and adrenals, which showed abnormalities during necropsy of dead animals and those sacrificed in extremis, were excised and macroscopically examined to determine the presence or absence of abnormalities. Ovaries were fixed in Bouin's solution (0.1M phosphate-buffered 10% formalin solution for long-term storage), while other organs and tissues were fixed in 0.1M phosphate-buffered 10% formalin solution for storage. Tissues with abnormalities were processed to prepare paraffin sections according to the conventional method and stained with hematoxylin-eosin to prepare specimens for histopathological examinations. The pituitary glands, thyroids, ovaries, uterus, cervix and vagina from all females in the control group and the high dose group, as well as females found to be infertile in other groups, were also processed to prepare specimens for histopathological examinations. Specimens for histopathological examinations were also prepared for the kidneys from all females because abnormalities were observed during necropsy in animals sacrificed in extremis in the mid-dose group.

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All live pups were sacrificed by ether inhalation and necropsied on day 21 after birth. Organs with abnormalities were fixed in 0.1M phosphate-buffered 10% formalin solution for storage.

Statistics:

The incidence of estrous cycles by type, copulation index, fertility index and incidence of morphological abnormalities in newborns were analyzed by Fischer’s exact test.
Findings of histopathological examinations were compared between the control group and each of the ADC groups: graded data by Mann-Whitney’s U-test and sums of positive grades by Fisher’s one-sided exact test.
All other data were first analyzed by Bartlett’s method with respect to the uniformity of variance in each group using data obtained from each animal and means determined for each litter as one sample.
If the variance was uniform, one-way analysis of variance was conducted.
If significant inter-group differences were found, multiple comparison was conducted by Dunnett’s method.
Kruskal-Wallis’ rank test was conducted if the variance was 0 in any group or if the variance was not uniform.
If significant inter-group differences were found, multiple comparison was made by the Dunnett-type test.

Reproductive indices:

Fertility index
Number of pairing days until copulation
Number of vaginal estruses during the mating period

Offspring viability indices:

Delivery index [(number of newborns/number of implantations) x 100],
Birth index [(number of live newborns/number of implantations) x 100]
Live birth index [(number of live newborns/number of newborns) x 100].
Sex ratio on day 0 after birth [(number of male live newborns/number of live newborns) x 100]
Viability index on day 4 after birth [(number of live pups on day 4 before adjustment of the litter size/number of live newborns on day 0 after birth) x 100]
Sex ratio on day 4 after birth [(number of male live pups on day 4 before adjustment of the litter size/number of live pups on day 4 before adjustment of the litter size) x 100]
Weaning index [(number of live pups on day 21 of lactation/number of live pups on day 4 after adjustment of the litter size) x 100]

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For details, see attached information below.

Mortality:

mortality observed, treatment-related

Description (incidence):

The following animals died: one from the control group (death on day 47 of treatment) and 5 from the 1,000 mg/kg group (on days 6, 12, 12, 41 and 69). None of these animals showed any abnormality until death except one from the 1,000 mg/kg group which showed crust formation. These deaths were thought to be attributable to errors in administration procedures because necropsy revealed the findings described below. Findings which were considered to be unrelated to errors in administration procedures were included in evaluation.

Animal No. MX1006: esophageal perforation and retention of oily pleural effusion
Animal No. MX04001: yellow soilage of perioral fur, test material-like yellow area mainly on the right lung lobes and yellow pigmentation on the tracheal lumen
Animal No. MX04003: esophageal perforation and retention of test material-like yellow, semi-transparent effusion in the thoracic cavity
Animal No. MX04004: hemorrhage-like dark red area in the esophagus around the hilar region and retention of a large volume of plural effusion with scattered yellow masses in the thoracic cavity
Animal No. MX04007: retention of a large volume of oily plural effusion and adhesion of yellow substances to the anterior diaphragm
Animal No. MX04023: dark red material in the esophagus and test material-like yellow material in the posterior lobe of the right lung

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: Body weight gain between days 92 and 99 was significantly greater (p<0.01) in the 300 mg/kg group than in the control group. However, no other significant differences were observed in body weight or body weight gain between the control group and each of the ADC groups.
Females: There were no significant differences in body weight or body weight gain between the control group and each of the ADC groups during the pre-mating, gestation or lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences between the control group and each of the ADC groups for males and females.

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Findings in males which died during the dosing period: No abnormalities were found in one animal each in the control and 1,000 mg/kg groups (MX01006 and MX04023). Of the other four males which died in the 1,000 mg/kg group, one (MX04001) showed atrophy of the seminiferous tubules associated with diffuse hyperplasia of Leydig cells in one testis. This animal also showed spermatic granuloma and multinucleated giant cells in the seminiferous tubules. The lumen of the epididymis on the affected side contained almost no sperm and was filled with cell debris. The ventral lobe of the prostate of this animal showed lymphocyte infiltration in the interstitium and neutrophil infiltration in the epithelium and lumen. Reproductive organs of the other three males which died before sexual maturation at an early stage of treatment (MX04003, MX04004 and MX04007) showed findings consistent with the age (5-6 weeks after birth). No abnormality was found. These animals were excluded from the histopathological evaluation of reproductive organs.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

In the observation of the estrous cycle during the 2-week pre-treatment period, one animal each in all groups, including the control group, showed monoestrus. After starting treatment, however, these animals showed normal estrous cycle. Before starting treatment, animals in the 100 mg/kg group and the 1,000 mg/kg group showed 5-day cycles, which were not seen in the control group. In the 1,000 mg/kg group, the mean number of days needed for return of estrus was slightly extended compared to the control group.
After starting treatment, the percentage of animals which showed changes in the estrous cycle showed no significant differences between the control group and any of the ADC groups. The mean number of days needed for return of estrus showed no significant changes in any ADC group, but significant differences (p<0.05) were noted between the 1000 mg/kg group and the control group.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

All animals copulated, but fertility was not confirmed due to the failure of females to become pregnant in the following animals: one male (MX01019) and one female (FB01019) in the control group, two males (MX02010 and MX02021) and two females (FB02010 and FB02021) in the 100 mg/kg group, three males (MX03003, MX03006 and MX03010) and three females (FB03003, FB03006 and FB03010) in the 300 mg/kg group, and one male (MX04009) and three females (FB04003, FB04004 and FB0009) in the 1000 mg/kg group. However, the fertility index, the number of pairing days until copulation, and the number of vaginal estruses during the mating period were comparable between the control group and each of the ADC groups.

Details are included in the attached document below.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects observed up to and including highest dose level tested (1000 mg/kg bw/day)

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Treatment with 1000 mg/kg to females caused death during the lactation period due to pelvic nephritis-like lesions.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the control group, all newborns showed decreases in temperature of the body surface on day 0 after birth, and all pups from one litter (dam No.: FB01017) which did not show any milk spot died on day 1 after birth. Pups from the ADC groups showed abnormalities due to poor lactation activities associated with worsening of general signs and symptoms in F0 females. In the 100 mg/kg group, all pups from one litter (dam No.: FB02005) showed no milk spot on day 1 after birth and died following decreases in temperature of the body surface the following day. Pups from another litter (dam No.: FB02019) showed no milk in the stomach on day 3 after birth. Temperature of the body surface decreased and milk spot disappeared the following day. All these pups showed emaciation thereafter and died by day 7 after birth. In the 300 mg/kg group, one litter (dam No.: FB03004) showed no milk spot on day 1 after birth. All pups from this litter died the following day following decreases in temperature of the body surface. In the 1,000 mg/kg group, pups from two litters (dam Nos.: FB04014 and FB04001) showed emaciation when the F0 females died on days 3 and 14 after birth. The former litter showed no milk spot. Another litter from the 1,000 mg/kg group (dam No.: FB04013) showed no milk spot on day 4 after birth. Milk spots became unclearly visible the following day and reappeared one day later. However, pups showed emaciation on day 12 after birth, followed by decreases in motor activity on day 14 after birth, and all died the following day. In addition, external malformations of the tail were suspected in some pups from two litters (dam Nos.: FB02002 and FB02025) in the 100 mg/kg group and one litter (dam No.: FB03009) from the 300 mg/kg group. However, this disappeared in the 300 mg/kg group as pups grew up. Other pups showed no abnormality.

Mortality / viability:

no mortality observed

Description (incidence and severity):

All of the four dead newborns found on day 0 after birth in the control group were stillbirths, while in the 100, 300, and 1,000 mg/kg groups, 12/17, 13/15, and 3/6 newborns found dead were judged to be stillbirths, respectively. The incidence of stillbirths was not significantly different between the control group and each of the ADC groups.
As mentioned above, all pups from some litters died in all groups including the control group. However, the number of live newborns, delivery index, number of live pups on days 0, 4 and 21 after birth, birth index, live birth index, viability of pups on day 4 after birth showed no significant differences between the control group and each of the ADC groups. The weaning index showed significant decreases only in the 100 mg/kg group (p<0.05).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of male and female pups showed no significant differences between the control group and each of the ADC groups at any time point.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy of dead pups, excess pups culled in the adjustment of the litter size, and live pups sacrificed on day 21 after birth showed various morphological changes including variations and malformations in pups from one to 4 litters per group in all groups including the control group. External malformations observed included a kinked tail in one pup (excess pup on day 4 after birth) from one litter in the control group (dam No.: FB01018). In the 100 mg/kg group, one (dead) pup from one litter (dam No.: FB02002) showed short trunk, club foot and brachyury. Excess pups from the same litter culled on day 4 after birth showed kinked tail (one pup), rudimentary tail (one pup) and brachyury/kinked tail (two pups). One pup from this litter sacrificed on day 21 after birth showed brachyury/kinked tail. One excess pup from another litter (dam No.: FB02025) culled on day 4 after birth showed kinked tail. No external malformation was observed in the 300 mg/kg group. In the 1,000 mg/kg group, excess pups from one litter (dam No.: FB04002) culled on day 4 after birth showed anal atresia/anury (one pup) and kinked tail (another pup). Two excess pups from another litter (dam No.: FB04019) culled on day 4 after birth showed kinked tail. Visceral malformations observed in the 100 mg/kg group included a horseshoe kidney and bifid apex observed in the dead pup from dam No. FB02002 which showed external malformations such as short trunk, as well as hydronephrosis observed in one pup from another litter (dam No.: FB02008) sacrificed on day 21 after birth. In addition, dilatation of the renal pelvis, a visceral variation, was observed in all groups including the control group. The total number of pups with morphological changes was counted, and the percentage of pups with these changes was compared between the control group and each of the ADC groups. The percentage of pups with external changes increased significantly in the 100 mg/kg group compared to the control group (p<0.05), but no significant inter-group differences were noted with respect to the percentage of pups with visceral changes.

Morphological examinations of live pups showed one pup with external malformations (short trunk, rudimentary tail, anal atresia and club foot) from one litter (dam No.: FB02002) in the 100 mg/kg group. This pup showed no milk spot. Six other pups from this litter had short tails. One pup from another litter (dam No.: FB02004) showed a bite in the rostral area. In the 300 mg/kg group, one pup from one litter (dam No.: FB03013) showed cyanosis. In the 1,000 mg/kg group, one pup from one litter (dam No.: FB04002) showed external malformations (anury and anal atresia). One pup from another litter (dam No.: FB04013) showed a loss of the tail tip due to trauma. One litter from the control group (dam No.: FB01017) showed no abnormality, but all pups from this litter showed decreases in temperature of the body surface without milk spot.

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

The sex ratio was comparable between the control group and each of the ADC groups at all time points.

VIABILITY (OFFSPRING)
All of the four dead newborns found on day 0 after birth in the control group were stillbirths, while in the 100, 300, and 1,000 mg/kg groups, 12/17, 13/15, and 3/6 newborns found dead were judged to be stillbirths, respectively. The incidence of stillbirths was not significantly different between the control group and each of the ADC groups.
As mentioned above, all pups from some litters died in all groups including the control group. However, the number of live newborns, delivery index, number of live pups on days 0, 4 and 21 after birth, birth index, live birth index, viability of pups on day 4 after birth showed no significant differences between the control group and each of the ADC groups. The weaning index showed significant decreases only in the 100 mg/kg group (p<0.05). The sex ratio was comparable between the control group and each of the ADC groups at all time points.

CLINICAL SIGNS (OFFSPRING)
In the control group, all newborns showed decreases in temperature of the body surface on day 0 after birth, and all pups from one litter (dam No.: FB01017) which did not show any milk spot died on day 1 after birth. Pups from the ADC groups showed abnormalities due to poor lactation activities associated with worsening of general signs and symptoms in F0 females. In the 100 mg/kg group, all pups from one litter (dam No.: FB02005) showed no milk spot on day 1 after birth and died following decreases in temperature of the body surface the following day. Pups from another litter (dam No.: FB02019) showed no milk in the stomach on day 3 after birth. Temperature of the body surface decreased and milk spot disappeared the following day. All these pups showed emaciation thereafter and died by day 7 after birth. In the 300 mg/kg group, one litter (dam No.: FB03004) showed no milk spot on day 1 after birth. All pups from this litter died the following day following decreases in temperature of the body surface. In the 1,000 mg/kg group, pups from two litters (dam Nos.: FB04014 and FB04001) showed emaciation when the F0 females died on days 3 and 14 after birth. The former litter showed no milk spot. Another litter from the 1,000 mg/kg group (dam No.: FB04013) showed no milk spot on day 4 after birth. Milk spots became unclearly visible the following day and reappeared one day later. However, pups showed emaciation on day 12 after birth, followed by decreases in motor activity on day 14 after birth, and all died the following day. In addition, external malformations of the tail were suspected in some pups from two litters (dam Nos.: FB02002 and FB02025) in the 100 mg/kg group and one litter (dam No.: FB03009) from the 300 mg/kg group. However, malformations disappeared in the 300 mg/kg group as pups grew up. Other pups showed no abnormality.

BODY WEIGHT (OFFSPRING): Body weight of male and female pups showed no significant differences between the control group and each of the ADC groups at any time point.

GROSS PATHOLOGY (OFFSPRING)
Necropsy of dead pups, excess pups culled in the adjustment of the litter size, and live pups sacrificed on day 21 after birth showed various morphological changes including variations and malformations in pups from one to 4 litters per group in all groups including the control group. External malformations observed included a kinked tail in one pup (excess pup on day 4 after birth) from one litter in the control group (dam No.: FB01018). In the 100 mg/kg group, one (dead) pup from one litter (dam No.: FB02002) showed short trunk, club foot and brachyury. Excess pups from the same litter culled on day 4 after birth showed kinked tail (one pup), rudimentary tail (one pup) and brachyury/kinked tail (two pups). One pup from this litter sacrificed on day 21 after birth showed brachyury/kinked tail. One excess pup from another litter (dam No.: FB02025) culled on day 4 after birth showed kinked tail. No external malformation was observed in the 300 mg/kg group. In the 1,000 mg/kg group, excess pups from one litter (dam No.: FB04002) culled on day 4 after birth showed anal atresia/anury (one pup) and kinked tail (another pup). Two excess pups from another litter (dam No.: FB04019) culled on day 4 after birth showed kinked tail. Visceral malformations observed in the 100 mg/kg group included a horseshoe kidney and bifid apex observed in the dead pup from dam No. FB02002 which showed external malformations such as short trunk, as well as hydronephrosis observed in one pup from another litter (dam No.: FB02008) sacrificed on day 21 after birth. In addition, dilatation of the renal pelvis, a visceral variation, was observed in all groups including the control group. The total number of pups with morphological changes was counted, and the percentage of pups with these changes was compared between the control group and each of the ADC groups. The percentage of pups with external changes increased significantly in the 100 mg/kg group compared to the control group (p<0.05), but no significant inter-group differences were noted with respect to the percentage of pups with visceral changes.

OTHER FINDINGS (OFFSPRING)
Morphological examinations of live pups showed one pup with external malformations (short trunk, rudimentary tail, anal atresia and club foot) from one litter (dam No.: FB02002) in the 100 mg/kg group. This pup showed no milk spot. Six other pups from this litter had short tails. One pup from another litter (dam No.: FB02004) showed a bite in the rostral area. In the 300 mg/kg group, one pup from one litter (dam No.: FB03013) showed cyanosis. In the 1,000 mg/kg group, one pup from one litter (dam No.: FB04002) showed external malformations (anury and anal atresia). One pup from another litter (dam No.: FB04013) showed a loss of the tail tip due to trauma. One litter from the control group (dam No.: FB01017) showed no abnormality, but all pups from this litter showed decreases in temperature of the body surface without milk spot.

Dose descriptor:

NOAEL

Generation:

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on F1 up to and including highest dose level tested (1000 mg/kg bw/day)

Critical effects observed:

Reproductive effects observed:

Conclusions:

Under the present conditions, NOELs for reproductive toxicity of 1,1'azobisformamide are considered to be 1000 mg/kg/day for males and females, and those for toxicity other than reproduction are considered to be 1000 mg/kg/day for males and 300 mg/kg/day for females.

Executive summary:

One-generation reproduction toxicity was investigated for 1,1 '-azobisformamide by daily oral administration of 0, 100, 300 and 1000 mg/kg to 5 week-old male and to 10 week-old female Sprague-Dawley strain (Crj:CD) rats (25 animals each sex/group). The test substance was given to the males for 10 weeks before mating and thereafter in total for 14 weeks, and to the females for 2 weeks before mating, and throughout the mating, gestation and lactation periods, thus in total 8 weeks.

The treatment did not affect their reproductive ability, including the estrous cycle, mating, parturition of the animals and viability, growth and morphology of offspring. While no adverse effects of 1,1 '-azobisformamide were found in males, treatment with 1000 mg/kg to females caused death during the lactation period due to pelvic pyelonephritic kidney lesions.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: chronic
Species:: rat
Quality of whole database:: Two reliable studies are available. The one-generation study (key study) has a Klimisch score 1, the three-generation study has a Klimisch score 2.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Short description of key information:

In a one-generation study realised according to the OECD guideline 415 and in compliance with GLP, the NOEL in rat by oral route has been found to be 1000 mg/kg bw/d (highest dose tested) for fertility and effects on reproduction in parental animals and for effects in F1 generation.

Effects on developmental toxicity

Description of key information

Two prenatal toxicity studies are available, one in rats and one in rabbits. The results of these studies indicate that ADCA does not exhibit developmental toxicity.

Link to relevant study records

Referenceopen all close all

Reference 1

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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

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.

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.

Dose / conc.:

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

Dose / conc.:

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

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.

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.

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.

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):

Early or late resorptions:

no effects observed

Description (incidence and severity):

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:

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

Key result

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).

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)

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

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.

Reference 2

Endpoint:

developmental toxicity

Remarks:

Rat

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental start date was 08 Jun 2020, and the experimental completion date was
22 Feb 2021. In life phase from 26th July 2020 to 13 August 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).

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
On 21 Jul 2020 and 23 Jul 2020, time-mated female Wistar Han Rats were received from
Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or
Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were
11-15 weeks old and weighed between 179 and 274 g at the initiation of dosing.
A health inspection was performed upon receipt of the animals.

Animal Identification
Before the first data collection on Day 2 post-coitum, each animal was identified using a
subcutaneously implanted electronic identification 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.

Selection, Assignment, Replacement, and Disposition of Animals
At arrival, animals were assigned to groups at random.

Husbandry

Housing
On arrival and following randomization, females were housed individually in Makrolon
plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15,
JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water
bottles. The room 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 18 to 24°C with a relative target humidity of 40 to 70% were
maintained. The actual daily mean temperature during the study period was 21 to 22°C with
an actual daily mean relative humidity of 49 to 74%. The values that were outside the targeted
range occurred for three consecutive days with a maximum of 76% and 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
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was
provided ad libitum throughout the study, except during designated procedures.
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.

Water
Municipal tap water was freely available to each animal via water bottles.
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 and nesting material, animals were provided
with paper and aspen wooden sticks, except when interrupted by study procedures/activities.
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; however, no examinations
or treatments were required.

In life phase from 26th July 2020 to 13 August 2020.

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.

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition,
and stability for the test item. The characterization of the test item was conducted in an
ISO quality environment. A Certificate of Analysis was provided to 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 by vortexing and magnetic
stirring. The dosing formulations were prepared at least weekly as a suspension, filled out in
daily portions and stored in the refrigerator. The dosing formulations were removed from the
refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed
within 24 hours after removal from the refrigerator.
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 or vehicle and no correction was
made for the purity/composition of the test item.
Details of the preparation and dispensing of the test item have been retained in the Study
Records.
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 (27-Jul-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 Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. 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 6 to Day 20 post-coitum, inclusive.
The dose volume for each animal was based on the most recent body weight measurement.
The doses were given using a plastic feeding tube.
The dosing formulations were stirred continuously during dose administration.
Dose pot identification via Provantis was used as additional check to verify the dosing
procedure according to Standard Operating Procedures.

Frequency of treatment:

Once daily

Duration of test:

Dosing for 15 days - Day 6 to Day 20 post-coitum, inclusive.

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22 (twenty-two)

Control animals:

yes, concurrent vehicle

Details on study design:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted 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.
This Study Plan was reviewed and agreed by the Animal Welfare Body of Charles River
Laboratories Den Bosch B.V. within the framework of Appendix 2 of project license
AVD2360020172866 approved by the Central Authority for Scientific Procedures on
Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

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 (Test Facility
Reference No. 20235613) and in an attempt to produce graded responses to
the test item. In this Dose Range Finder, no effects on body weight (gain) and (relative) food
consumption were observed up to 1000 mg/kg/day. Clinical signs included yellow colored
feces for two females at 1000 mg/kg/day on Days 11-13 post-coitum. Macroscopy did not
reveal any alterations.

Maternal examinations:

Mortality/Moribundity
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.

Clinical Observations
Cage side observations were performed once daily, starting on Day 6 post-coitum onwards up to the day prior to necropsy. During the dosing period, this observation was performed postdose. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Cage debris was examined to detect premature birth.

Detailed Clinical Observations
The animals were removed from the cage, and a detailed clinical observation was performed weekly, beginning during the Pre-Treatment Period, and on the day of necropsy.

Body Weights
Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

Food Consumption
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

Water Consumption
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 (22 post-coitum): Necropsy; collection of gross lesions; organ weights. Histology and histopathology examinations wee also conducted on the thyroid gland of all animals.

Necropsy
All animals 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). No organs (except for the gravid uterus and thyroid gland) were weighed.

Necropsy procedures were performed by qualified personnel with appropriate training and
experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably
qualified person, was available.

Organ Weights
The thyroid and uterus were weighed at necropsy for all animals. Paired organs were weighed
together. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated.

Tissue Collection and Preservation
The thyroid gland and macroscopic abnormalities were collected from all animals and
preserved in 10% neutral buffered formalin.

Histology
Thyroid glands of all animals were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Histopathology
Thyroid glands of all animals were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Ovaries and uterine content:

Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the uterus.
- The number of implantation sites.
- The number and distribution of live and dead fetuses.
- The number and distribution of early and late resorptions.
- The sex of each fetus based on the anogenital distance.
For one female at 1000 mg/kg bw/day no macroscopically visible implantation sites were present, the nongravid uteri was stained using the Salewski technique in order to detect any
former implantation sites.

Blood sampling:

Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected from the jugular vein randomized between 7:00 and 9:00 a.m. in the animal facility. After collection, samples were transferred to the appropriate laboratory for processing.

Thyroid Hormone
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant.
Blood samples were processed for serum, and serum was analyzed for the parameters listed:
Triiodothyronine (T3)
Thyroxine (T4)
Thyroid-Stimulating Hormone (TSH)

After clotting and centrifugation, serum was used for measurement of T3 (Schaijk), T4 and
TSH (‘s Hertogenbosch).

Bioanalysis Triiodothyronine (T3) via LC-MS Method
Serum samples were analyzed for T3 according to the bioanalytical method validated in Test Facility Study No. 20213516.
General data concerning chemicals, reagents, preparation of stock solutions, calibration standards and quality control samples (QCs) as well as the analytical conditions was stored
under Test Facility Study No. 20235614.

Fetal examinations:

Method of Euthanasia
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube.

Fetal Examinations
Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations, as described in the following sections. 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 sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.

Visceral Examinations
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were 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 this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination 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.

Skeletal Examinations
All fetuses were eviscerated, followed by fixation in 96% aqueous ethanol, and maceration in potassium hydroxide. Thereafter, they were stained with Alizarin Red S by a method similar to that described by Dawson.
Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with
heads). 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; Biochemistry variables; Organ weights; relative organ weights to body; 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.

Indices:

Maternal Variables

Body Weight Gains: Calculated for the following intervals: Days 6 to 9,
9 to 12, 12 to 15, 15 to 18, 18 to 21, and 6 to 21 post-coitum.
Corrected Body Weight Gain: Body weight on Day 21 post-coitum - body weight
on Day 6 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:

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. Limited HCD is provided where skeletal malformations were observed. This was obtained from 6882 fetus/1290 litters, time period not specified.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period.
Any clinical signs noted during the treatment period (i.e. salivation, fur loss and scabs)
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 with the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

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.
One female dosed at 100 mg/kg bw/day was observed with a low body weight gain during the
complete treatment period. This could be explained by the uterine content of this female, as
only two live fetuses were present at scheduled necropsy. As this only occurred in a single
dam at the low dose group, this was unrelated to treatment with the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Decreased serum levels of thyroid stimulating hormone (TSH) were noted at 100, 300 and 1000 mg/kg bw/day (0.74x, 0.81x and 0.82x of controls, respectively), however without reaching statistical significance and in the absence of a dose response. This difference in TSH was therefore considered not to be test item-related.
Serum levels of total triiodothyronine (Total T3) and total thyroxine (Total T4) were considered to be unaffected by treatment up to 1000 mg/kg bw/day.
The results are attached below in tabular form.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in thyroid gland weights.
Thyroid weights and thyroid:body weight ratios of treated animals were considered to be
similar to those of control animals. The results are attached below in tabular form.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to
have arisen as a result of treatment with the test item.
All of the recorded macroscopic findings were within the range of background gross
observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations in the thyroid glands.
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.

Number of abortions:

no effects observed

Description (incidence and severity):

All results are attached below in tabular form.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

All females, except for Female No. 67 (1000 mg/kg/day), were pregnant and had litters with
viable fetuses only.
The percentage of postimplantation loss was statistically significantly increased at
300 mg/kg/day compared to concurrent control, which was caused by the higher mean
number of early resorptions at this dose level. However, as values remained within historical
control data and no dose response was observed, this was considered to be unrelated to
treatment with the test item.
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control
and test groups were similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The percentage of postimplantation loss was statistically significantly increased at
300 mg/kg/day compared to concurrent control, which was caused by the higher mean
number of early resorptions at this dose level. However, as values remained within historical
control data and no dose response was observed, this was considered to be unrelated to
treatment with the test item.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Fetuses were delivered by caesarian section on post-coital day 21, therefore changes to pregnancy duration

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

No maternal toxicity was observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Tested up to 1000 mg/kg bw/day

Key result

Abnormalities:

no effects observed

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 1000 mg/kg/day. The results are attached below in tabular form.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

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

Changes in sex ratio:

no effects observed

Description (incidence and severity):

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

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.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no test item-related effects on fetal anogenital distance (both sexes) noted after
treatment up to 1000 mg/kg/day.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no external malformations and variations in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on skeletal morphology following treatment up to
1000 mg/kg/day.
Skeletal malformations occurred in two fetuses at 1000 mg/kg/day and in one control fetus.
The high dose fetuses either had fused mandibles with a single incisor socket (68-R7) or
severely fused sternebrae and absent cervical arch (69-L3). The single occurrence of these
two fetuses with malformations affecting different areas did not suggest any association with
the test item.
The affected control fetus had bent humeri (12-R10) which was as such considered to be of
spontaneous origin.
Among the less severe skeletal variations, incidences of supernumerary cervical short ribs at
1000 mg/kg/day were higher than in the other groups. However, because statistical
significance was not achieved and incidences remained below the historical control maximum
values (see below), the presence of these ribs at the high dose was considered not test item-related.
Other skeletal variations occurred in the absence of a dose-related incidence trend and/or
infrequently. Therefore, they were considered not test item-related.
HCD Rat (CRL:WI(Han)) – Total No. Fetuses/Litters Examined Skeletally n = 6882/1290
7th Cervical Rudimentary Rib(s) / Ossification Site(s); mean (min-max): 4.2 (0.0-9.9)
Fetal%: 4.16 (0.00-9.65)
Litter%: 17.6 (0.0-40.9)

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on visceral morphology following treatment up to
1000 mg/kg/day.
Visceral malformations did not occur in any group.
The observed visceral variations affected the liver (supernumerary lobe) and ureter
(convoluted). These findings occurred infrequently and did not suggest any association with
the test item at any dose.

Details on embryotoxic / teratogenic effects:

No test substance related effects were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Tested up to 1000 mg/kg bw/day

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

Treatment related:

Conclusions:

Based on the results of this prenatal developmental toxicity study the maternal and
developmental No Observed Adverse Effect Level (NOAEL) for Azodicarbonamide Unifoam
AZ VI-50 in rats were established as being at least 1000 mg/kg bw/day.

Executive summary:

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 Wistar Han rats from Day 6 to 20 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, 100, 300, 1000 mg/kg/day, based on the results of the Dose Range Finder (Test Facility Reference No. 20235613).

The study design was as follows:

Group No	Test Item	Dose level mg/kg/day	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Females
1	-	0(control)	5	0	22
2	Azodicarbonamide Unifoam AZ VI-50	100	5	20	22
3		300	5	60	22
4		1000	5	200	22

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 500 mg/kg bw/day
Study duration:: subacute
Species:: rabbit
Quality of whole database:: Two reliable studies are available (both Klimisch 1 studies).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

The rats were dosed up to and including 1000 mg/kg bw/day, no adverse effects were observed on the mothers or on the pups. In the rabbit study the highest dose level tested (500 mg/kg bw/day) resulted in significant systemic toxicity to the mothers (including mortality), however no adverse effects were observed on the development of the pups. As the NOAEL in rabbits is lower than the NOAEL in rats, the rabbit study is chosen as the key study.

Justification for classification or non-classification

Based on the current data-set, the substance is not classified for reproductive and/or developmental toxicity according to Regulation (EC) No 1272/2008.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

NET GAINS (g) AT 12 WEEKS OF FOUR GENERATIONS OF RATS
Group	Diet code	F0		F1		F2		F3
Group	Diet code	25M	25F	10M	10F	10M	10F	10M	10F
A	Control (forB, C,D)	290	175	262	154	270	158	253	133
B	100X HADA	280	174	317	185	314	185	295	166
C	316X HADA	281	179	326	184	269	168	240	136
D	1000X HADA	291	183	288	162	306	173	303	152
E	10X ADA	273	172	307	184	280	157	284	169
A2	Control (for E)	276	171	314	171	298	171	283	160

NET GAINS (g) AT 12 WEEKS OF FOUR GENERATIONS OF RATS
Group	Diet code	F0		F1		F2		F3
Group	Diet code	25M	25F	10M	10F	10M	10F	10M	10F
A	Control (forB, C,D)	290	175	262	154	270	158	253	133
B	100X HADA	280	174	317	185	314	185	295	166
C	316X HADA	281	179	326	184	269	168	240	136
D	1000X HADA	291	183	288	162	306	173	303	152
E	10X ADA	273	172	307	184	280	157	284	169
A2	Control (for E)	276	171	314	171	298	171	283	160

	Generation	A Control for B, C, D	B 100 X HADA	C 316X HADA	D 1000X HADA	E 10X ADA	A2 Control for E
Average number pupsper litter
At birth	F0	8.3	7.8	9.2	9.7	8.7	8.8
	F1	9.5	9.9	9.8	9.7	10.1	10
	F2	9.3	8.4	7.8	9.1	9.5	9.4
At weaning	F0	6.5	6.8	7.8	7.6	7.5	8
	F1	7.2	7.9	8.6	8.3	8.3	8.2
	F2	6.6	6	6.7	7.9	7.9	8
Average weight of pups at 21 days (g)	F0	49.5	47.3	50.2	50.8	44.3	44.5
	F1	45.0	49.0	44	46.7	47.7	46.3
	F2	47.1	47;4	46.4	45.5	40.0	44.2
Fertility index	F0	57	64	72	40	80	88
	F1	95	90	70	85	65	90
	F2	90	80	95	90	90	95
Gestation index	F0	100	100	100	100	94	96
	F1	95	94	100	100	100	100
	F2	100	94	100	100	95	100
Viability index	F0	68	70	67	84	86	84
	F1	62	80	83	82	73	80
	F2	60	60	72	82	85	84
Lactation index	F0	89	89	85	88	85	84
	F1	88	95	96	92	96	91
	F2	93	99	99	99	98	96

a Fertility index (pregnancies/matings) x100
b Gestation index (litters born/pregnancies) x 100
c Viability index (pups surviving at 4 days/pups born) x 100
d Lactation index (pups weaned/pups at 4 days) x 100

Registration Dossier

Registration Dossier

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Diss Factsheets

C,C'-azodi(formamide)

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Reference 3

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

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