Number range CHAPTER

**Evaluation of the Toxicology Database**

**On**

**Azadioxabicycooctane**

FOR

REREGISTRATION ELIGIBILITY DECISION (RED)

September 28, 2005

1.  **HAZARD CHARACTERIZATION**

Azadioxabicyclooctane consists of an equilibrium mixture of three
chemicals (**I:**
5-hydroxymethoxymethyl-1-aza-3,7-dioxabicyclo(3,3,0)octane; **II:**
5-hydroxymethyl-1-aza-3,7-dioxabicyclo(3,3,0)octane; **III:**
5-hydroxypoly(methylene-oxy)methyl-1-aza-3,7-dioxabicyclo(3,3,0)octane).
These cannot be divided into components for individual testing. This
mixture is registered for control of slime-forming bacteria and fungi at
the following use sites: oil recovery drilling muds and packer fluids;
industrial adhesives and coatings; resin/latex/polymer emulsions;
metalworking cutting fluids; latex paints; paper/paper products;
specialty industrial products; and wet-end additives for industrial
processing chemicals

Azadioxabicyclooctane has a moderate order of acute toxicity via the
oral and dermal routes of exposure (Toxicity Category III) and a
moderate to severe acute toxicity via the inhalation route (Toxicity
Category II). Azadioxabicyclooctane produces severe irritation to the
eyes (Toxicity Category I) and moderate irritation to the skin (Toxicity
Category III).

Repeated dose toxicity data for azadioxabicyclooctane are limited to a
90-day oral toxicity study in the rat, a 21-day dermal toxicity study in
the rabbit, and a dermal developmental toxicity study in the rat.

In a 90-day oral toxicity study (MRID 41641606 reformat of 00109245,
supplemental information 410801010), Nuosept® 95 (Lot No. 005-267) was
administered to 20 CD1 Sprague-Dawley derived rats/sex/dose in drinking
water at dose levels of 0, 125, 625, or 3125 ppm (equivalent to 0, 20,
100, or 500 mg/kg bw/day; actual intake reported was 0, 13, 62, and 245
mg/kg/day). Four rats/sex/group were kept for a 4-week recovery period
to determine reversibility of effects. No signs of toxicity due to
Nuosept 95 treatment were noted in mortality, clinical observations,
hematology, clinical chemistry, ophthalmology, gross pathology, or
histopathology. Body weight and body weight gain were lower in high-dose
males and females compared to their respective controls. Although food
consumption was reduced in the intermediate and high-dose groups in both
males and females, the food efficiency was unaffected. Water consumption
was drastically (approximately 30-50%) reduced in high-dose males and
females throughout the entire study period. Due to the reduction in
water consumption, the intake of test article did not reach predicted
levels. Based on the analytical concentration of the test article in the
water and the water intake data, the daily doses were estimated to be
10.6, 56.5, and 228.9 mg/kg/day in males and 12.2, 64.3, and 255.9
mg/kg/day for females. The LOAEL is 56.5 and 64.3 mg/kg/day in males and
females, respectively, based on the reduction in water consumption. The
NOAEL is 10.6 and 12.2 mg/kg/day for males and females, respectively.
The 90-day study is classified **unacceptable/guideline** but is
**upgradable**.

In a 21-day dermal toxicity study (MRID 41641605), Nuosept® 95/Nuosept®
C (Lot number and purity not reported) was administered directly to the
skin of New Zealand White rabbits (5/sex/group) at doses of 0, 100, 300,
and 1000 mg/kg/day, 6 hours/day for 5 days/week during a 21-day period.
No treatment-related effects on clinical observations and mean absolute
or relative organ weights were observed. Significant findings were
presented for food consumption, body weight, hematology, and clinical
chemistry parameters, however these findings were not considered to be
treatment-related. Extensive damage to the treated skin was observed in
all of the rabbits in the high-dose group. Numerous microscopic changes
in the skin as well as increases of neutrophils and platelets were
observed and were considered to be secondary to tissue repair. The
dermal LOAEL is 100 mg/kg/day (based on severe dermal effects). The
dermal NOAEL for Nuosept 95/Nuosept C could not be determined because
mild skin effects were observed at the low dose. The systemic NOAEL is
greater than or equal to 1000 mg/kg/day and the LOAEL is greater than
1000 mg/kg/day. This 21-day dermal toxicity study in the rabbit is
classified as **acceptable (non-guideline)**.

In a dermal developmental toxicity study (MRID 41537501, reformat of
41699001), Nuosept® 95 (Batch No. 006-097-597, 50.38%;) in distilled
water was applied dermally to mated female Crl:COBS CD (SD) BR VAF^+^
rats (25/dose) at dose levels of 0, 100, 300, or 1000 mg/kg/day from
days 6 through 15 of gestation (gd 6-15). An additional 25 mated female
rats were administered distilled water from gd 6-15. There were no
treatment related effects in mortality, clinical signs, weight gain,
food consumption, gross necropsy parameters and cesarean parameters.
There was clear evidence of maternal dermal effects in all treatment
groups. The low-dose group had well-defined erythema, scabbing that
obscured some of the treatment site, and well-defined edema in most
animals by study termination. The intermediate-dose group displayed
moderate/well-defined erythema. Half of the animals had scabbing that
completely obscured the treatment site, and edema that was well-defined
by study termination. The high-dose group exhibited well-defined
erythema, scabbing that obscured the treatment site in all animals by gd
10, and moderate to severe edema that extended to the underlying
musculature. The LOAEL for maternal dermal toxicity is 100 mg/kg/day
(based on severe dermal irritation); a NOAEL could not be established.
The systemic maternal toxicity NOAEL is greater than or equal to 1000
mg/kg/day; the systemic maternal toxicity LOAEL is greater than 1000
mg/kg/day. There were no other embryotoxic or fetotoxic effects observed
in this study. The developmental NOAEL is greater than or equal to
1000 mg/kg/day; the developmental LOAEL is greater than 1000 mg/kg/day.
This developmental toxicity study in the rabbit is classified as
**unacceptable/ guideline** and is **upgradable.**

There are no data on the reproductive toxicity of azadioxabicyclooctane.
There are also no carcinogenicity nor chronic toxicity data for
azadioxabicyclooctane.

Azadioxabicyclooctane has been tested for mutagenic activity in several
assays, including a bacterial DNA damage/repair assay (MRID 93050019;
93050020), dominant lethal assay (MRID 00088953), unscheduled DNA
synthesis assay (MRID 93050021; 42711001; 41642401), micronucleus assay
(41728601), *in vitro* cell transformation assay (MRID 93050022), and
the Ames assay (MRID 93050017; 93050018). In the Ames Salmonella test ,
azadioxabicyclooctane was negative in all strains tested, but
deficiencies in the studies were identified and these deficiencies must
be addressed to upgrade the studies. Azadioxabicyclooctane was also
negative in the micronucleus assay, but was positive in one unscheduled
DNA synthesis assay (93050021) and the bacterial DNA damage/repair
assay. Several tests (MRID numbers 93050022 through 93050025) were
conducted with C3H/10T^1/2^ cell cultures examining cell transformation
*in vitro*. Although results were largely negative, these studies
collectively did not properly identify the test material being examined.
In fact, only one study (MRID 93050018) properly characterized the test
material. Thus, although the data suggest largely negative responses,
the lack of test article characterization (especially in light of
positive responses observed in two studies) points to the need for
proper test article characterization before an adequate conclusion can
be made about the mutagenicity of azadioxabicyclooctane.

From the available repeat dose toxicity studies, there was no evidence
of neurotoxicity of azadioxabicyclooctane.

2.  **REQUIREMENTS**

Insert Table

3.  **DATA GAPS**

The ADTC determined that there are several data gaps in the hazard
database for azadioxabicyclooctane based on the current use patterns.
Due to the potential for inhalation exposure in the occupational
setting, a repeated dose (90-day) inhalation toxicity study should be
performed with azadioxabicyclooctane. The oral study used for selection
of the inhalation endpoint does not adequately characterize potential
inhalation hazard and was used with added uncertainty factors (10x) in
the absence of inhalation data. A route-specific study would reduce or
eliminate this uncertainty. An acceptable dermal sensitization study
must also be submitted.

To address data requirements for indirect food uses of antimicrobials,
the Agency has established an interim two-tiered system for toxicology
testing requirements in order to complete a risk assessment for indirect
food uses of antimicrobial pesticide chemicals for which the FDA has
established a food additive regulation that specifically states that the
use is \"safe\", but for which the Agency requires its own assessment.
Tier I toxicology data requirements would apply to all indirect food
additives that result in residue concentrations in food ranging from
0-200ppb. The requirements would consist of an acute toxicity testing
battery, a subchronic toxicity study in the rodent, a developmental
toxicity study in the rodent (rat), and a mutagenicity testing battery.
Each of these data requirements has been fulfilled for AZA. The Agency
also conducts a literature search and can also conduct a Structure
Activity Relationship analysis (SAR) if appropriate.

Tier II studies would be triggered by the presence of significant (i.e.
=200ppb) residues in food or evidence of significant toxicity from the
Tier I data set, which may include developmental / reproductive, or
other systemic toxicity such as presence of neoplastic growth or
significant target organ toxicity. In such cases, chronic toxicity and
carcinogenicity testing would be required in addition to the above Tier
I studies.

For AZA, the Agency will hold in reserve a two-generation reproduction
toxicity study in the rat and a subchronic toxicity study in the
non-rodent which would become data requirements pending the evaluation
of the Tier 1 data to adequately address risk from indirect food uses of
azadioxabicyclooctane

Separately, risks from the metalworking fluid use of
azadioxabicyclooctane cannot be adequately characterized without conduct
of chronic toxicity studies in the rat and mouse as well as
carcinogenicity studies in these two species. Metalworking fluid use is
considered a high exposure antimicrobial use pattern and requires
chronic toxicity and carcinogenicity data.

**4.0 HAZARD ASSESSMENT**

1.  **Acute Toxicity**

[Adequacy of database for Acute Toxicity]{.underline}: The acute
toxicity database for azadioxabicyclooctane is considered incomplete. An
acceptable dermal sensitization study must be submitted.

The acute oral toxicity of azadioxabicyclooctane is low, with a
calculated median lethal dose of 1940 mg/kg from a study in the rat
(MRID 41641601). Pale kidneys, enlarged spleen, and livers with focal
hemosiderosis were observed in this study. The acute dermal toxicity of
azadioxabicyclooctane is also low, with a dermal median lethal dose of
greater than 2000 mg/kg (MRID 41671801). Dermal effects were noted, but
no systemic toxicity. The acute inhalation toxicity study (MRID
42650901) showed a median lethal dose range of between 0.441 mg/L and
0.819 mg/L, with epistaxis, labored breathing, rales, and rhinorrhea in
all dose groups. Corneal opacity was observed in the primary eye
irritation study (MRID 41641602) resulting in a Toxicity Category I
classification. Moderate dermal irritation effects were noted in the
primary dermal irritation study (MRID 41641603), leading to a Toxicity
category III classification.

The acute toxicity data for azadioxabicyclooctane is summarized in Table
1.

+--------+-------------------+-------------+-----------------+--------+
| **Ta   |                   |             |                 |        |
| ble 1. |                   |             |                 |        |
| Acute  |                   |             |                 |        |
| To     |                   |             |                 |        |
| xicity |                   |             |                 |        |
| P      |                   |             |                 |        |
| rofile |                   |             |                 |        |
| for    |                   |             |                 |        |
| Azadi  |                   |             |                 |        |
| oxabic |                   |             |                 |        |
| yclooc |                   |             |                 |        |
| tane** |                   |             |                 |        |
+========+===================+=============+=================+========+
| **Gui  | **Study Type/**   | **MRID      | **Results**     | **To   |
| deline |                   | Number/**   |                 | xicity |
| Nu     | **Test substance  |             |                 | Cate   |
| mber** | (% a.i.)**        | *           |                 | gory** |
|        |                   | *Citation** |                 |        |
+--------+-------------------+-------------+-----------------+--------+
| 87     | Acute Oral- Rat   | MRID        | LD~50~ = 1940   | III    |
| 0.1100 | Nuosept® 95 (50%  | 41641601    | mg/kg/day       |        |
|        | a.i.)             |             |                 |        |
| (      |                   |             |                 |        |
| §81-1) |                   |             |                 |        |
+--------+-------------------+-------------+-----------------+--------+
| 87     | Acute Dermal-     | MRID        | LD~50~ \> 2000  | III    |
| 0.1200 | Rabbit            | 41671801    | mg / kg         |        |
|        |                   |             |                 |        |
| (      | Nuosept® 95 (50%  |             |                 |        |
| §81-2) | a.i.)             |             |                 |        |
+--------+-------------------+-------------+-----------------+--------+
| 87     | Acute Inhalation- | MRID        | LC~50~  0.441  | II     |
| 0.1300 | Rat               | 42650901    | mg/L            |        |
|        |                   |             |                 |        |
| (      | Nuosept® 95 (50%  |             |                 |        |
| §81-3) | a.i.)             |             |                 |        |
+--------+-------------------+-------------+-----------------+--------+
| 87     | Primary Eye       | MRID        | Corrosive       | I      |
| 0.2400 | Irritation-       | 41641602    |                 |        |
|        | Rabbit            |             |                 |        |
| (      |                   |             |                 |        |
| §81-4) | Nuosept® 95 (50%  |             |                 |        |
|        | a.i.)             |             |                 |        |
+--------+-------------------+-------------+-----------------+--------+
| 87     | Primary Dermal    | MRID        | Moderate        | III    |
| 0.2500 | Irritation-       | 41641603    | irritant        |        |
|        | Rabbit            |             |                 |        |
| (      |                   |             |                 |        |
| §81-5) | Nuosept® 95 (50%  |             |                 |        |
|        | a.i.)             |             |                 |        |
+--------+-------------------+-------------+-----------------+--------+
| 87     | Dermal            | NA          | Assumed         | no     |
| 0.2600 | Sensitization     |             | Sensitizer      | data   |
|        |                   |             |                 | ava    |
| (      |                   |             |                 | ilable |
| §81-6) |                   |             |                 |        |
+--------+-------------------+-------------+-----------------+--------+

**4.2 Subchronic Toxicity**

[Adequacy of database for Subchronic Toxicity]{.underline}: Subchronic
toxicity data for azadioxabicyclooctane are limited to a 90-day oral
toxicity study in the rat and a 21-day dermal toxicity study in the rat.

**870.3100 90-day Oral Toxicity Study (rat)**

In a 90-day oral toxicity study (MRID 41641606 reformat of 00109245,
supplemental information 410801010), Nuosept® 95 (Lot No. 005-267) was
administered to 20 CD1 Sprague-Dawley derived rats/sex/dose in drinking
water at dose levels of 0, 125, 625, or 3125 ppm (equivalent to 0, 20,
100, or 500 mg/kg bw/day; actual intake reported was 0, 13, 62, and 245
mg/kg/day). Four rats/sex/group were kept for a 4-week recovery period
to determine reversibility of effects.

No signs of toxicity due to Nuosept 95 treatment were noted in
mortality, clinical observations, hematology, clinical chemistry,
ophthalmology, gross pathology, or histopathology. Body weight and body
weight gain were lower in high-dose males and females compared to their
respective controls. Although food consumption was reduced in the
intermediate and high-dose groups in both males and females, the food
efficiency was unaffected. Water consumption was drastically
(approximately 30-50%) reduced in high-dose males and females throughout
the entire study period. The water consumption in these high-dose rats
recovered after the test article was removed from the drinking water.
Water consumption in the intermediate-dose group was slightly
(approximately 10-20%) lower than controls with statistical significance
reached sporadically. These rats resumed their consumption of water
during the recovery period. Due to the reduction in water consumption,
the intake of test article did not reach predicted levels. Based on the
analytical concentration of the test article in the water and the water
intake data, the daily doses were estimated to be 0, 10.6, 56.5, and
228.9 mg/kg/day in males and 0, 12.2, 64.3, and 255.9 mg/kg/day for
females.

**The LOAEL is 56.5 and 64.3 mg/kg/day in males and females,
respectively, based on the reduction in water consumption. The NOAEL is
10.6 and 12.2 mg/kg/day for males and females, respectively.**

The 90-day study is classified **unacceptable/guideline** but is
**upgradable.** If the histopathology report is reformatted and
re-submitted for review, conclusions regarding organ weight changes
and/or enzyme changes can be made when an understandable histopathology
report is submitted. There were several deficiencies in the submitted
histopathology report: (1) several animal numbers were referenced for
which there were no histopathology reports (animal #'s 430 and higher);
(2) incidence of lesions in control animals that appeared higher than in
treated animals, such as individual cell necrosis of the liver,
hemosiderosis of the spleen, and chronic respiratory disease. If an
acceptable report is re-submitted, the study may then be considered for
upgrade. This can be done, as the week 4 results for hematology can be
discarded based on faulty methodology (guideline requires only a 13 week
measurement), and a recovery group is not part of the guideline
requirement, therefore all the deficiencies from that examination can be
disregarded and the conclusions set aside for that group. Information on
the characterization of the test article also needs to be provided.
Azadioxabicyclooctane is an equilibrium mixture of three isomers, and
information needs to be provided on the percentages of these three
isomers in the mixture used in this study.

**21-day Dermal Toxicity Study**

In a 21-day dermal toxicity study (MRID 41641605), Nuosept® 95/Nuosept®
C (Lot number and purity not reported) was administered directly to the
skin of New Zealand White rabbits (5/sex/group) at doses of 0, 100, 300,
and 1000 mg/kg/day, 6 hours/day for 5 days/week during a 21-day period.

No treatment-related effects on clinical observations and mean absolute
or relative organ weights were observed. Significant findings were
presented for food consumption, body weight, hematology, and clinical
chemistry parameters. These findings were not considered to be
treatment-related, however, because of the following reasons: (1) body
weights and food consumption were reduced by week 3 due to the extensive
skin damage and decreased mobility in the high-dose group; (2)
statistical changes occurred sporadically; and (3) a dose-dependent
relationship was not observed. Extensive damage to the treated skin was
observed in all of the rabbits in the high-dose group. Numerous
microscopic changes in the skin as well as increases of neutrophils and
platelets were observed and were considered to be secondary to tissue
repair.

**The dermal LOAEL is 100 mg/kg/day (based on severe dermal effects).
The dermal NOAEL for Nuosept 95/Nuosept C could not be determined
because skin effects were observed at the low dose. The systemic NOAEL
is greater than or equal to 1000 mg/kg/day. The systemic LOAEL is
greater than 1000 mg/kg/day.**

This 21-day dermal toxicity study in the rabbit is classified
**acceptable (non-guideline)**. Although the study does not satisfy the
guideline requirement for a 21/28-day dermal toxicity study (OPPTS
870.3200 \[82-2\]; OECD 410), no systemic effects were observed rats
exposed to Nuosept95/Nuosept C at concentrations up to and including a
limit dose. Information needs to be provided on the composition of the
test article. Azadioxabicyclooctane is an equilibrium mixture of three
isomers and information should be provided on the percentages of these
three isomers in the mixture. To date, the registrant has not supplied
this information.

**4.3 Prenatal Developmental Toxicity**

[Adequacy of database for Prenatal Developmental Toxicity]{.underline}:
There is only one developmental toxicity study (MRID 41537501, reformat
of 41699001) in rats available for this chemical conducted by the dermal
route.

**Developmental Toxicity -- Rat**

In a developmental toxicity study (MRID 41537501, reformat of 41699001),
Nuosept 95 (Batch No. 006-097-597, 50.38% a.i.) in distilled water was
applied as a neat material to mated female Crl:COBS CD (SD) BR VAF^+^
rats (25/dose) at dose levels of 0, 100, 300, or 1000 mg/kg/day from
days 6 through 15 of gestation (gd 6-15). An additional 25 mated female
rats were administered distilled water from gd 6-15.

There were no treatment-related effects in mortality, food consumption,
or cesarean parameters. There was clear evidence of maternal dermal
effects in all treatment groups. The low-dose group had well-defined
erythema and scabbing that obscured some of the treatment site, and
well-defined edema in most animals by study termination. The
intermediate-dose group displayed moderate/well-defined erythema. Half
of the animals had scabbing that completely obscured the treatment site,
and edema that was well-defined by study termination. The high-dose
group exhibited well-defined erythema, scabbing that obscured the
treatment site in all animals by gd 10, and moderate to severe edema
that extended to the underlying musculature.

Systemic effects were shown by a slight dose-related reduction in mean
bodyweight gain statistically lower than controls in the 1000 mg/kg/day
group (p\<0.5) during the gd 7-14 period. Enlargement of lymph nodes
were observed at autopsy in the 300 (1 animal) and 1000 (2 animals)
mg/kg/day dose groups. There were no adverse effects on litter
parameters or embryofoetal development.

**The LOAEL for maternal dermal toxicity is 100 mg/kg/day (based on
dermal irritation); a NOAEL could not be established. The systemic
maternal toxicity NOAEL is 300 mg/kg/day; the maternal toxicity LOAEL is
1000 mg/kg/day (based on decreased body weight gain). The developmental
NOAEL is greater than or equal to 1000 mg/kg/day; the developmental
LOAEL is greater than 1000 mg/kg/day.**

This developmental toxicity study in the rabbit is classified as
**unacceptable/guideline** and is **upgradable**. The study report
contains several minor deficiencies, including lack of information on
the homogeneity, concentration, or stability of the test material in the
vehicle and historical control data. However, there is useful
information concerning the dermal toxicity of the chemical in this
study, and testing up to a dermal limit dose showed no systemic effects.
Although the dosing duration was not considered long enough for a dermal
developmental toxicity study (dosing should begin on day 0 for dermal
studies), the lack of a systemic effect at the limit dose is supported
by the submitted 21/28 day dermal toxicity study, which also found no
systemic effects up to and including a limit dose.

**4.4 Reproductive Toxicity**

[Adequacy of database for Reproductive Toxicity]{.underline}: There are
no data on the reproductive toxicity of azadioxabicyclooctane. For the
indirect food use of this chemical, this study is being held in reserve.

**4.5 Chronic Toxicity**

[Adequacy of database for Chronic Toxicity]{.underline}: There are no
data on chronic toxicity of azadioxabicyclooctane. Metalworking cutting
fluid use triggers the requirement for chronic toxicity studies as
described on the 1987 Antimicrobial Data Call In Notice.

**4.6 Carcinogenicity**

[Adequacy of database for Carcinogenicity]{.underline}: A determination
of carcinogenic potential cannot be made based on the available data.
Metalworking cutting fluid use triggers the requirement for
carcinogenicity studies as described on the 1987 Antimicrobial Data
Call-In Notice.

7.  **Mutagenicity**

Azadioxabicyclooctane has been tested for mutagenic activity in several
assays, including a bacterial DNA damage/repair assay (MRID 93050019;
93050020), dominant lethal assay (MRID 00088953), unscheduled DNA
synthesis assay (MRID 93050021; 42711001; 41642401), micronucleus assay
(41728601), *in vitro* cell transformation assay (MRID 93050022), and
the Ames assay (MRID 93050017; 93050018). In the Ames Salmonella test,
azadioxabicyclooctane was negative in all strains tested, but
deficiencies in the studies were identified and these deficiencies must
be addressed to upgrade the studies. Azadioxabicyclooctane was also
negative in the micronucleus assay, but was positive in one unscheduled
DNA synthesis assay (93050021) and the bacterial DNA damage/repair
assay. Several tests (MRID numbers 93050022 through 93050025) were
conducted with C3H/10T^1/2^ cell cultures examining cell transformation
*in vitro*. Although results were largely negative, these studies
collectively did not properly identify the test material being examined.
In fact, only one study (MRID 93050018) properly characterized the test
material. Thus, although the data suggest largely negative responses,
the lack of test article characterization (especially in light of
positive responses observed in two studies) points to the need for
proper test article characterization before an adequate conclusion can
be made about the mutagenicity of azadioxabicyclooctane.

[*Salmonella/* Mammalian Activation Gene Mutation;]{.underline} OPPTS
870.5265 \[§84-2\] In a reverse gene mutation assay in bacteria (MRID
93050017 reformat of 00088956), strains TA98, TA100, TA1535, TA1537 and
TA1538 of *S. typhimurium* were exposed to Nuosept® 95 (Lot No. 005-267)
in water at concentrations of 0, 0.01, 0.05, 0.23, 0.45 and 0.9 μL/plate
in the presence and absence of mammalian metabolic activation (S9-mix).
The S9-fraction was obtained from Aroclor 1254 induced male
Sprague-Dawley rat liver.

Nuosept 95 was tested up to cytotoxic concentrations. A preliminary
assay with TA100 showed no cytotoxicity at concentrations up to 0.29
μL/plate and virtually complete cell killing at the 0.94 μL/plate and
higher concentrations. The highest concentration tested in the
mutagenicity assay, 0.9 μL/plate was cytotoxic as evidenced by a
reduction in the number of revertants per plate in all strains compared
to the solvent controls. At no Nuosept 95 concentration, with or without
S9-mix, was there at least a doubling of the mutant frequency in any
tester strain over the solvent control values. The greatest increase was
seen in strain TA100 where the mutant frequency at 0.45 μL/plate, both
with and without S9-mix, was approximately 1.9 times the solvent control
value. The positive and solvent controls induced the appropriate
responses in the strains in which they were tested. **There was no
evidence of induced mutant colonies over background.**

This study is classified as **unacceptable/guideline** due to incomplete
positive control data. In the absence of S9-mix, only strains TA1535 and
TA1537 were treated with a positive control and in the presence of
S9-mix, only strains TA98, TA100 and TA1538 were treated with the
positive control.

[*Salmonella/* Mammalian Activation Gene Mutation]{.underline}; OPPTS
870.5265 \[§84-2\] In a reverse gene mutation assay in bacteria (MRID
93050018 reformat of 00088974), strains TA98, TA100, TA1535, TA1537 and
TA1538 of *S. typhimurium* were exposed to Nuosept® 95 (Lot No. 005-267)
in water at concentrations of 0, 5, 10, 50, 100 and 250 μg/plate in an
initial assay and to concentrations of 0, 375, 500, 625 and 750 μg/plate
in a second assay. Testing was in the presence and absence of mammalian
metabolic activation (S9-mix) from Aroclor 1254 induced rat liver.

Nuosept 95 was tested up to cytotoxic concentrations. Toxicity, as
evidenced by changes in the background lawn of bacteria, was seen in all
tester strains at 750 μg/plate in the absence of S9-mix and in TA1535 in
the presence of S9-mix. Toxicity was seen in the absence of S9-mix at
500 μg/plate in TA98 and TA1538 and at 625 μg/plate in all strains
except TA1535. The mitotic index (number of revertants on Nuosept 95
treated plates/number of revertants on solvent control plates) remained
below two in all tester strains at all concentrations, with or without
S9-mix. In almost all cases the mitotic index was 1.0 or lower. The
positive controls induced the appropriate responses in the presence of
S9-mix. **There was no evidence of induced mutant colonies over
background.**

This study is classified as **unacceptable/guideline.** Few experimental
details were provided and positive controls were not used in
non-activated tests. In addition, the composition of the S9-mix was not
provided.

[*In Vitro* Mammalian Cytogenics - Micronucleus Assay in Mouse Bone
Marrow Polychromatic Erythrocytes]{.underline} OPPTS 870.5395 \[§84-2\]
In a Swiss-Webster mouse bone marrow micronucleus assay (MRID 41728601),
five mice/sex/dose were treated by oral gavage (two treatments separated
by 24 hours) with Nuosept® 95 (Lot No. 129593, purity not reported) at
doses of 0, 300, 600 and 1200 mg/kg body weight in males and 0, 600,
1200 and 2400 mg/kg body weight in females. Bone marrow cells were
harvested at 24 and 48 hours post-treatment. The vehicle was sterile
deionized water.

There were signs of toxicity during the study. A preliminary toxicity
assay was used to determine doses used in the micronucleus assay. In the
micronucleus assay, all mice in the solvent control and the 300 or 600
mg/kg Nuosept 95 dose groups appeared normal. One male in the 1200 mg/kg
group had rough fur, a humped back and labored breathing on day 2 and
was found dead on day 3. Nine of a total of 17 females dosed at 2400
mg/kg had rough fur and humped backs with four also showing labored
breathing. Four females from the 2400 mg/kg dose groups were found dead
on day 3 and three others had rough fur and humped backs. Other clinical
signs seen in the 2400 mg/kg groups were lacrimation from both eyes and
closed eyes. The solvent controls showed a frequency of micronucleated
PCEs at 24 and 48 hours of 0.14% and 0.16%, respectively, in males and
0.14% and 0.12%, respectively, in females. The frequency of
micronucleated PCEs in Nuosept 95 treated mice did not exceed 0.20% in
males or 0.16% in females at any concentration at either harvest time.
No increase was statistically significant. The urethane positive control
produced a frequency of micronucleated PCEs in males at 24 and 48 hours
of 2.59% and 1.16%, respectively. These increases were statistically
significant (p\<0.05). No positive control was used in females. Positive
and solvent control values were acceptable. **There was no significant
increase in the frequency of micronucleated polychromatic erythrocytes
in bone marrow PCEs in either sex at any tested dose at either harvest
time.**

This study is classified as **unacceptable/guideline** and
**upgradable**. The test substance purity and composition is required.

[Rat Dominant Lethal Mutagenicity Assay]{.underline}; OPPTS 870.5450
\[§84-2\] In a CD1 Sprague-Dawley rat dominant lethal assay (MRIDs
240259303 and 93050026, reformat of 00088953), male rats were treated
for 10 consecutive weeks with Nuosept® 95 (Lot No. 005-267) in their
drinking water at doses of 0, 125, 625 or 3125 pom of active ingredient
(estimated daily average for the 10 week period of 0, 14, 66 and 268
mg/kg body weight/day of Nuosept 95 active ingredients). After the 10
week dosing period, each male was mated with two untreated females per
week for two weeks.

Nuosept 95 was tested to a dose limited by taste aversion. Average daily
water consumption was comparable in the solvent control and two lower
dose Nuosept 95 groups; however, the overall average daily water
consumption for the 3125 pom group was approximately 69% of the solvent
control group. The average weight gain of the rats over the 10 week
dosing period was 280 g for the solvent control, 255 g for the positive
control and 251, 284 and 244 g for the 125, 625 and 3125 pom Nuosept 95
dosed groups, respectively. The lower weight gain was considered a
result of reduced water intake rather than a toxic effect of the test
material. No other clinical signs were reported. All treated mice
survived until the end of the study. The females were sacrificed 16 days
after the first day of cohabitation and examined for pregnancy, living
fetuses, early and late fetal death and corpora lutea and for
intercurrent infections. The following parameters were determined: total
implants per pregnant female (live fetuses plus early and late fetal
deaths), dead implants per pregnant female (early and late fetal
deaths), total number of dead implants per total implants, corpora lutea
per pregnant female, live implants per pregnant female and
preimplantation loss (the difference between total corpora lutea and
total implants), the number of Corpora lutea, implantations, living
embryos and fetuses, and dead implantations (sum of the deciduomata,
resorptions and dead embryos and fetuses). Pre- and post-implantation
losses were determined. With one exception, there were no other
statistically significant differences between Nuosept 95 treated groups
and solvent controls in any of the measured parameters at any dose in
either the first or second week of mating. The one exception was a
statistically significant reduction in the average number of corpora
lutea per female in the 625 pom group in the second week of mating
compared to the solvent control (15.40 vs 17.53). This difference was
not considered biologically significant by the testing laboratory
because the count was within normal limits (which were not given) and
comparable to that of all groups in the first mating week. Positive and
solvent control values were appropriate. **There was no significant
difference between the control group and Nuosept 95-treated groups with
respect to fertilization rate, dead implantations, living implantations,
preimplantation loss or total implantations.**

This study is classified as **unacceptable/guideline**. The route of
administration of the test substance is insufficient. The only
statistically and biologically significant effect observed in the
high-dose group was reduced water consumption and resultant reduced body
weight. Because Nuosept 95 was administered in the drinking water, this
effect may be due to taste aversion of the test substance. If the test
substance had been administered by another method, such as oral gavage,
higher doses may have been evaluated.

[Bacterial Damage/Repair in *Salmonella typhimurium* Assay]{.underline};
OPPTS 870.5500 \[§84-2\] In a differential killing/growth inhibition
assay in bacteria (MRID 93050019 reformat of 00088957), strains WP2 (uvr
A^+^ rec A^+^) and WP100 (uvr A^-^ rec A^-^) of *Escherichia coli* and
strains TA 1978 (uvr B^+^) and TA 1538 (uvr B^-^) of *Salmonella
typhimurium* were exposed to Nuosept® 95 (Lot No. 005-267) in water in a
suspension assay at concentrations of 0.03, 0.3, 0.6 and 0.9 μL/mL in
the presence and absence of mammalian metabolic activation (S9-mix). The
S9-fraction was obtained from Aroclor 1254 induced male Sprague-Dawley
rat liver.

Nuosept 95 was tested up to cytotoxic concentrations. In the absence of
S9-mix, no cell killing was seen in either the repair proficient or
deficient *E. coli* strains at the lowest concentration tested, 0.03
μL/mL; however, survival of the repair deficient strains was virtually
zero at all higher concentrations compared to average survivals ranging
from 54% to 42% in the repair proficient strain. The *S. typhimurium*
strains were somewhat more sensitive with survivals at the lowest
concentration of 81% and 72% for the repair proficient and deficient
strains, respectively. Survival of the repair proficient strain ranged
from 31% to 18% at the higher concentrations and virtually complete
killing of the repair deficient strain at all higher concentrations.
Decreasing survival indices (defined as the percent survival of the
repair deficient strain by the percent survival of the repair proficient
strain) with increasing concentrations of test chemicals are considered
indicative of genotoxicity in this assay. In the presence of S9-mix, the
results for the *E. coli* strains were similar to those in the absence
of S9-mix. Survivals of the *S. typhimurium* strains at the two lowest
concentrations of Nuosept 95 were actually higher for the repair
deficient strain (72% and 92% for the repair proficient and deficient
strains at 0.03 μL/mL, respectively, and 20% and 36% for the repair
proficient and deficient strains at 0.3 μL/mL, respectively). At 0.6
μL/mL, survival of the repair deficient strain was 15% compared to 28%
for the repair proficient strain with comparable values at 0.9 μL/mL of
11% and 23%. Only the 0.6 and 0.9 µL/mL concentrations presented
positive results in the suspension assay (\< 0.8). The negative, solvent
and positive controls induced the appropriate responses in the
corresponding strains. **There was evidence of greater growth inhibition
or cell killing in repair-defective strains compared to repair competent
strains.**

This study is classified as **acceptable/guideline.**

[Bacterial Damage/Repair in *Escherichia coli* and *Salmonella
typhimurium* Mutagenicity Assay]{.underline} PPTS 870.5500 \[§84-2\] In
a differential killing/growth inhibition assay in bacteria (MRID
93050020 reformat of 00088958), strains W3110 (pol A^+^) and p3478 (pol
A^-^) of *Escherichia. coli* and *Salmonella typhimurium* strains TA
1978 (uvr B^+^) and TA 1538 (uvr B^-^) were exposed to Nuosept® 95 (Lot
No. 005-267) in sterile distilled water at concentrations of 50, 100,
500, 1000 and 5000 μg/mL in suspension culture in the presence and
absence of mammalian metabolic activation (S9-mix). The two *E. coli*
strains were also exposed to Nuosept 95 in a disk assay (10 μL of
Nuosept 95 on a paper disk in the center of the plate) but the
activation status was not given. The S9-fraction was obtained from
Aroclor 1254 induced rat liver.

Nuosept 95 was tested up to cytotoxic concentrations. In the disk assay,
the diameter of the zone of inhibition (scored as a ratio of the zone of
inhibition and of the pol A^-^ to the zone of inhibition of the pol
A^+^) of Nuosept 95 treated strains was 36 mm for the pol A^+^ strain
and 43 mm for the pol A^-^ strain compared to 0.0 mm for the solvent
control in both strains. A chemical is considered to have a positive
response if the difference in the zones of inhibition is greater than or
equal to 3 mm. The difference of 7 mm in Nuosept® 95 treated bacteria
was equal to the MNNG positive control difference (28 and 35 mm for the
pol A^+^ and pol A^-^ strains, respectively). In the suspension assay,
the testing laboratory's criterion for a positive response, a survival
index (ratio of survival of repair deficient/repair proficient strains)
of less than 0.8, was met at 100, 500, 1000, and 5000 μg/mL in both
tester pairs with and without S9-mix with the exception of a negative
response at 100 μg/mL in the presence of activation. The assay was
negative for all strains with and without metabolic activation at the 50
μg/mL concentration. The survival of all strains (except *E. coli* (pol
A^+^) only reduces to 37.3% at 5000 μg/mL with S9-mix) was reduced to
zero at 5000 μg/mL. Positive and solvent control values were acceptable.
Few experimental details were provided with this study. **The number of
replicate plates and/or cultures was not given, individual plate counts
were not provided (just summary data), the S9-mix composition was not
given, and the positive control for the *S. typhimurium* strains without
S9-mix was not specified. There was evidence of greater growth
inhibition or cell killing in repair-defective strains compared to
repair competent strains.**

This study is classified as **unacceptable/guideline** and
**upgradable**. Information on individual plate counts and the S9-mix
composition are required.

[Unscheduled DNA Synthesis in Primary Rat Hepatocytes/ Mammalian Cell
Cultures Mutagenicity Assay]{.underline} OPPTS 870.5550 \[§84-2\] In an
unscheduled DNA synthesis assay (MRID 93050021 reformat of 00088959),
primary rat hepatocyte cultures from male F-344 rats were exposed to
Nuosept® 95 (lot number and purity not provided) in culture medium at
concentrations of 0, 0.0156, 0.0316, 0.0625, 0.125, 0.25, 1.0 and 2.0
μL/mL for one hour.

Nuosept 95 was tested up to cytotoxic concentrations. Concentrations,
ranging from 0.0058 to 47.6 μL/mL, were tested in a preliminary
cytotoxicity assay (trypan blue exclusion) with no viable cells
remaining in cultures exposed to 1.49 μL/mL and higher concentrations. A
concentration of 0.74 μL/mL reduced viability to approximately 45% of
the negative control at two hours post-treatment and to zero when
incubation was allowed to continue for 24 hours post-treatment. Two UDS
assays were performed. Results from the first assay were considered
unusable due to poor cell morphology and distribution on the coverslips
and an abnormally low positive control value. The data from the first
assay were not reported. In the second UDS assay, Nuosept 95
concentrations of 0.5 μL/mL and higher were quite toxic with no
surviving cells 24 hours after treatment. Concentrations of 0.0156 to
0.25 μL/mL gave a range of survivals at 24 hours post-treatment from
100% to 29.1%. The average percentage of nuclei with six or more grains
was 32.3% and 25.9% at Nuosept 95 concentrations of 0.0625 and 0.125
μL/mL, respectively, thus exceeding the criterion of 21% required for a
positive response as established for this particular assay. The average
percentage of nuclei with six or more grains at 0.25 μL/mL was 18.0%,
not quite reaching the level for a positive response; however,
cytotoxicity was high at this concentration (29.1% survival at 24 hours)
and the variation between the three cultures at this dose was said to
range from 8% to 36% (individual culture data were not presented).
Cytotoxicity may have inhibited UDS activity at this dose. The average
percentage of nuclei with  6 grains in the negative (culture medium)
and positive (2-AAF) controls were 11.0% and 84.7%, respectively. The
controls induced the appropriate response. **There was evidence of
unscheduled DNA synthesis, as determined by radioactive tracer
procedures \[nuclear silver grain counts\].**

This study is classified as **unacceptable/guideline** and
**upgradable**. The test substance purity and composition is required.

[Unscheduled DNA Synthesis in Primary Rat Hepatocytes/ Mammalian Cell
Cultures]{.underline}; OPPTS 870.5550 \[§84-2\]. In an *ex vivo*
unscheduled DNA synthesis (UDS) assay (MRID 42711001), three male
Fischer-344 rats per dose group were given a single oral dose (gavage)
each of Nuosept® 95 (lot No. 05249587, purity not provided) in water at
concentrations of 0, 875, 1750 or 3500 mg/kg body weight. Rats were
sacrificed two or 16 hours after treatment and hepatocytes isolated and
analyzed in culture for induced UDS activity. The percentage of
hepatocytes in repair was also determined.

Nuosept 95 was tested to toxic levels (approximately 20, 40 and 80% of
the estimated LD~50~ of 4401 mg/kg calculated from the results of two
preliminary toxicity assays). Rats in the 16-hour 1750 and 3500 mg/kg
dose groups had rough fur on the morning after dosing and one rat in the
16-hour 3500 mg/kg dose group died before the scheduled sacrifice time.
Viability of the isolated hepatocytes averaged approximately 80% in all
Nuosept 95 treated groups and in the solvent control group and
approximately 70% in the positive control group. No increase in the mean
net nuclear grain count over the solvent control value of -7.4 was seen
at any tested Nuosept 95 concentration at either the two-hour or 16-hour
sacrifice time. The mean percentage of cells in repair did not exceed 1%
(the solvent control value) at any tested Nuosept 95 concentration at
either sacrifice time. The positive control (dimethylnitrosamine)
induced a mean net nuclear grain count of 18.3 and a percentage of cells
in repair of 25.6%. The positive and solvent controls induced the
appropriate responses. **There was no evidence that unscheduled DNA
synthesis, as determined by radioactive tracer procedures (nuclear
silver grain counts) was induced by Nuosept 95 as tested in this
study.**

This study is classified as **unacceptable/guideline** and
**upgradable**. The test substance purity and composition is required.

[*In Vitro* Cell Transformation Assay in C3H/10T½ Cells]{.underline}
Other Genotoxicity \[§84-4\] In an *in vitro* cell transformation assay
(MRID 93050022 reformat of 00088960), C3H/10T½ cell cultures were
exposed without metabolic activation to Nuosept® 95 (Lot No. 005-267) at
concentrations of 0, 0.0005, 0.001, 0.002, and 0.004 μl/ml for 18 hours.
After replacement of the medium and further incubation for 5 weeks with
weekly refeeding, cultures were fixed, stained, and scored for
morphological transformation. Foci of transformed cells were observed in
5 of 12 cultures. No dose response was observed but that is not required
for this assay. Cytotoxicity was demonstrated by reduction of relative
cloning efficiency below 50% at the two higher doses. The positive and
solvent control values were appropriate.

This study is classified as **acceptable/non-guideline**.

[*In Vitro* Cell Transformation Assay in C3H/10T½ Cells]{.underline};
Other Genotoxicity \[§84-4\]. In an *in vitro* cell transformation assay
(MRID 93050023 reformat of 00088961), C3H/10T½ cell cultures were
exposed without metabolic activation to Nuosept® 95 (Lot No. 005-267) at
concentrations of 0, 0.0003, 0.001, and 0.003 μl/ml for 24 hours. After
replacement of the medium and further incubation for 4-6 week with twice
weekly refeeding, cultures were fixed, stained, and scored for
morphological transformation. No Type II or Type III foci of transformed
cells were observed in any treatment group. Cytotoxicity was evident at
the high dose, demonstrated by reduction of relative cloning efficiency
below 50%. Positive and solvent control values were appropriate.

This study is classified as **unacceptable/non-guideline** and
**upgradable** because it cannot be ascertained whether T1597 could have
been tested at a considerably higher dose and because the duration of
incubation of the cell cultures following treatment was not precisely
specified. If data can be presented to show that a significantly higher
dose would be too toxic (\>90% reduction of cloning efficiency) and to
specify the duration of post-treatment incubation of the cultures, the
study could be upgraded to acceptable.

[*In Vitro* Cell Transformation Assay in C3H/10T½ Cells]{.underline};
Other Genotoxicity \[§84-4\]. In an *in vitro* cell transformation assay
(MRID 93050024 reformat of 00088962), C3H/10T½ cell cultures were
exposed to R-1162 (Lot number and purity not reported) without metabolic
activation to R-1162 at concentrations of 0, 0.0098, 0.0195, 0.039, and
0.078 μl/ml for 18 hours. After replacement of the medium and further
incubation for 5 weeks with weekly refeeding, cultures were fixed,
stained, and scored for morphological transformation. Foci of
transformed cells were not observed at any dose level. The number of
transformed foci in the positive and solvent controls was appropriate.
Cytotoxicity was demonstrated by reduction of relative cloning
efficiency below 50% at the highest dose, although unusually low
survival in the solvent control cultures caused relative cloning
efficiencies to be higher than expected in the cell transformation
assay.

This study is classified as **unacceptable/non-guideline** and
**upgradable**. Information on the test substance lot number, purity,
and composition was not provided.

[*In Vitro* Cell Transformation Assay in C3H/10T½ Cells]{.underline};
Other Genotoxicity \[§84-4\]. In an *in vitro* cell transformation assay
(93050025 reformat of 00088963), C3H/10T½ cell cultures were exposed
without metabolic activation to R-1143 (Lot \# 11-20-1979) at
concentrations of 0, 0.0006, 0.0012, 0.0024, and 0.0048 μl/ml for 18
hours. After replacement of the medium and further incubation for 5
weeks with weekly refeeding, cultures were fixed, stained, and scored
for morphological transformation. Foci of transformed cells were not
observed at any dose level. The number of transformed foci for the
positive and solvent controls was appropriate. Cytotoxicity was
demonstrated by reduction of relative cloning efficiency below 50% at
the highest dose tested.

This study is classified as **unacceptable/non-guideline** and
**upgradable**. Information on the test substance lot number, purity,
and composition was not provided.

**4.8 Neurotoxicity**

[Adequacy of database for Neurotoxicity]{.underline}: From the available
repeat dose toxicity studies, there was no evidence of neurotoxicity of
azadioxabicyclooctane.

**4.9 Metabolism and Pharmacokinetics**

[Adequacy of database for Metabolism and Pharmacokinetics]{.underline}:
The database does not provide information on the metabolism and
pharmacokinetics of azadioxabicyclooctane.

**5.0 TOXICITY ENDPOINT SELECTION**

1.  **See Section 7.1, Summary of Toxicological Doses and Endpoint
    Selection, Table 3.**

2.  **Dermal Absorption**

[Dermal Absorption Factor]{.underline}: Since a dermal endpoint was
selected from a dermal toxicity study, a dermal absorption factor is not
needed for azadioxabicyclooctane.

3.  **Classification of Carcinogenic Potential**

There are no carcinogenicity data for azadioxabicyclooctane. A
determination of carcinogenic potential cannot be made based on the
available data. Metalworking cutting fluid use triggers the requirement
for carcinogenicity studies and chronic toxicity studies as described on
the 1987 Antimicrobial Data Call In Notice.

**6.0 FQPA CONSIDERATIONS**

**6.1 Reproductive Toxicity Study Conclusions**

There are no data on the reproductive toxicity of azadioxabicyclooctane.
For the indirect food use of this chemical, this study is being held in
reserve.

**6.2 Pre-and/or Postnatal Toxicity**

A.  [Determination of Susceptibility]{.underline}

The ADTC concluded that there is no evidence for susceptibility to
azadioxabicyclooctane from the available data, but the data are very
limited to one developmental toxicity study. For this reason, extra
uncertainty factors will be employed to account for the lack of a
complete database until more data are made available.

B.  [Degree of Concern Analysis and Residual Uncertainties]{.underline}

Uncertainties exist with respect to determination of both developmental
and reproductive effects of azadioxabicyclooctane, as the data are
limited to the one study mentioned above.

C.  [Proposed Hazard-based Special FQPA Safety Factor(s)]{.underline}:

The ADTC recommended that the special 10x hazard-based safety factor
under the FQPA be **retained (10x)** for azadioxabicyclooctane. There is
only one prenatal dermal developmental toxicity study available for
azadioxabicyclooctane, and there is no reproductive toxicity information
or studies. While the developmental study showed no evidence of
susceptibility of offspring to this chemical, the route of
administration is not a good indicator of potential ffects from oral
exposures. In addition, as noted above, information on the test article
characterization is not resolved at this time. The retention of 10x FQPA
hazard-based safety factor is consistent with the Agency's published
guidance.

(see <http://www.epa.gov/oppfead1/trac/science/determ.pdf)>

**6.3 Recommendation for a Developmental Neurotoxicity Study**

The ADTC reserved its recommendation for a developmental neurotoxicity
study, pending fulfillment of the data gaps for developmental and
reproductive toxicity.

**7.0 SUMMARY OF TOXICOLOGICAL DOSES AND ENDPOINTS FOR
AZADIOXABICYCLOOCTANE FOR USE IN HUMAN RISK**

1.  **Summary Table of Toxicological Dose and Endpoint Selection (Table
    2)**

+-------------+-------------+---------------+-------------------------+
| *           | **Dose Used | **Special     | **Study and             |
| *Exposure** | in Risk     | FQPA SF\* and | Toxicological Effects** |
|             | Assessment, | Level of      |                         |
| *           | UF**        | Concern for   |                         |
| *Scenario** |             | Risk          |                         |
|             |             | Assessment**  |                         |
+=============+=============+===============+=========================+
| **Acute     | **NOAEL =   | **FQPA SF =   | **90 day oral toxicity  |
| Dietary**   | 10.6        | 10x**         | in rats**               |
|             | mg/kg/day** |               |                         |
| **(gen.     |             | **aPAD =      | **LOAEL = 10.6          |
| pop.)**     | **UF =      | [acute        | mg/kg/day based on      |
|             | 100**       | RfD]{         | decreased water         |
|             |             | .underline}** | consumption at 56.5     |
|             |             |               | mg/kg/day in males.**   |
|             |             | **FQPA SF**   |                         |
|             |             |               |                         |
|             |             | **= 0.01      |                         |
|             |             | mg/kg/day**   |                         |
+-------------+-------------+---------------+-------------------------+
| **Acute     | **NOAEL =   | **FQPA SF =   | **90 day oral toxicity  |
| Dietary**   | 10.6        | 10x**         | in rats**               |
|             | mg/kg/day** |               |                         |
| **(females  |             | **aPAD =      | **LOAEL = 10.6          |
| 13+)**      | **UF =      | [acute        | mg/kg/day based on      |
|             | 100**       | RfD]{         | decreased water         |
|             |             | .underline}** | consumption at 56.5     |
|             |             |               | mg/kg/day in males.**   |
|             |             | **DB UF**     |                         |
|             |             |               |                         |
|             |             | **= 0.01      |                         |
|             |             | mg/kg/day**   |                         |
+-------------+-------------+---------------+-------------------------+
| **Chronic   | **NOAEL =   | **FQPA SF =   | **90 day oral toxicity  |
| Dietary**   | 10.6        | 10x**         | in rats**               |
|             | mg/kg/day** |               |                         |
| **(All      |             | **cPAD =**    | **LOAEL = 10.6          |
| pop         | **UF =      |               | mg/kg/day based on      |
| ulations)** | 300**       | **[chronic    | decreased water         |
|             |             | RfD]{         | consumption at 56.5     |
|             |             | .underline}** | mg/kg/day in males.**   |
|             |             |               |                         |
|             |             | **FQPA SF**   |                         |
|             |             |               |                         |
|             |             | **= 0.003     |                         |
|             |             | mg/kg/day**   |                         |
+-------------+-------------+---------------+-------------------------+
| **          |             |               | **No endpoint           |
| Short-Term/ |             |               | required**              |
| Interm      |             |               |                         |
| ediate-term |             |               |                         |
| Incidental  |             |               |                         |
| Oral (1-30  |             |               |                         |
| days; 30    |             |               |                         |
| days- 6     |             |               |                         |
| months)**   |             |               |                         |
+-------------+-------------+---------------+-------------------------+
| **Dermal    | **dermal    | *             | Co-critical studies:    |
| (all        | LOAEL=**    | *Occupational |                         |
| d           |             | MOE = 300 (   | **21-day dermal         |
| urations)** | **100       | ST and IT)**  | toxicity in rats**      |
|             | mg/kg/day** |               |                         |
|             |             | **= 1000      | LOAEL = 100 mg/kg/day   |
|             |             | (LT)**        | (severe dermal effects) |
|             |             |               |                         |
|             |             |               | **developmental         |
|             |             |               | toxicity in rats**      |
|             |             |               |                         |
|             |             |               | LOAEL = **100**         |
|             |             |               | mg/kg/day (severe       |
|             |             |               | dermal effects)         |
+-------------+-------------+---------------+-------------------------+
| *           | **90-day    | *             | **90 day oral toxicity  |
| *Inhalation | oral study  | *Occupational | in rats**               |
| (all        | NOAEL= 10.6 | MOE = 3000**  |                         |
| durations   | mg/kg/day** |               | **LOAEL = 10.6          |
| )**         |             |               | mg/kg/day based on      |
|             | **          |               | decreased water         |
|             | (inhalation |               | consumption at 56.5     |
|             | absorption  |               | mg/kg/day in males.**   |
|             | rate =      |               |                         |
|             | 100%)**     |               |                         |
+-------------+-------------+---------------+-------------------------+
| **Cancer    |             |               | **no cancer data        |
| (oral,      |             |               | available**             |
| dermal,     |             |               |                         |
| in          |             |               |                         |
| halation)** |             |               |                         |
+-------------+-------------+---------------+-------------------------+

UF = uncertainty factor, FQPA SF = Special FQPA safety factor, NOAEL =
no observed adverse effect level, LOAEL = lowest observed adverse effect
level, PAD = population adjusted dose (a = acute, c = chronic) RfD =
reference dose, MOE = margin of exposure, LOC = level of concern, NA =
Not Applicable

***NOTE:*** The Special FQPA Safety Factor recommended by the ADTC
**assumes** that the exposure databases (dietary food, drinking water,
and residential) are complete and that the risk assessment for each
potential exposure scenario includes all metabolites and/or degradates
of concern and does not underestimate the potential risk for infants and
children.

**8.0 TOXICITY PROFILE TABLES**

**8.1 Acute Toxicity Profile Table - (See Section 4.1, Acute Toxicity,
Table 1).**

**8.2 Subchronic, Chronic and Other Toxicity Profiles Table (Table 3)**

+-------------------+-------------------+------------------------------+
| **Guideline       | **MRID Number     | **Results**                  |
| Number/**         | (Year)/**         |                              |
|                   |                   |                              |
| **Study Type/**   | **Citation/**     |                              |
|                   |                   |                              |
| **Test Substance  | **Classification/ |                              |
| (% a.i.)**        | Doses**           |                              |
+===================+===================+==============================+
| 870.3100 (§82-1)  | 41641606,         | **Oral Toxicity (males)**    |
|                   | reformat of       |                              |
| 90-day Oral - Rat | 00109245,         | NOAEL = 10.6 mg/kg/day       |
|                   | supplemental      | (males)                      |
| Azad              | 410801010         |                              |
| ioxabicyclooctane |                   | = 12.2 mg/kg/day (females)   |
| purity 50%        | **Acceptable -    |                              |
|                   | Guideline**       | LOAEL = 56.5 mg/kg/day       |
|                   |                   | (males)                      |
|                   | 0, 10.6, 56.5, or |                              |
|                   | 228.9 mg/kg/day   | = 64.3 mg/kg/day (females)   |
|                   | (males)           |                              |
|                   |                   | based on reduced water       |
|                   | 0, 12.2, 64.3, or | consumption.                 |
|                   | 255.9 mg/kg/day   |                              |
|                   | (females)^1^      |                              |
+-------------------+-------------------+------------------------------+
| 870.3200 (§82-2)  | 41641605          | **Dermal Toxicity**          |
|                   |                   |                              |
| 21-day Dermal -   | **Acceptable -    | NOAEL not established        |
| Rabbit            | Non-guideline**   |                              |
|                   |                   | LOAEL = 100 mg/kg/day        |
| Azad              | 0, 100, 300, or   | (severe dermal effects)      |
| ioxabicyclooctane | 1000 mg/kg/day    |                              |
| purity not        |                   | **Systemic Toxicity**        |
| reported          |                   |                              |
|                   |                   | NOAEL ≥ 1000 mg/kg/day       |
|                   |                   | (highest dose tested)        |
|                   |                   |                              |
|                   |                   | LOAEL \> 1000 mg/kg/day (not |
|                   |                   | established)                 |
+-------------------+-------------------+------------------------------+
| 870.3700a (§83-3) | 41537501,         | **Dermal Maternal Toxicity** |
|                   | reformat of       |                              |
| Developmental -   | 41699001          | NOAEL not established        |
| Rat               |                   |                              |
|                   | **Acceptable -    | LOAEL = 100 mg/kg/day        |
| Azad              | Non-guideline**   | (severe dermal irritation)   |
| ioxabicyclooctane |                   |                              |
| purity 50.38%     | 0, 100, 300, or   | **Systemic Maternal          |
|                   | 1000 mg/kg/day    | Toxicity**                   |
|                   |                   |                              |
|                   |                   | NOAEL = 300 mg/kg/day        |
|                   |                   |                              |
|                   |                   | LOAEL = 1000 mg/kg/day       |
|                   |                   | (decreased body weight gain) |
|                   |                   |                              |
|                   |                   | **Developmental Toxicity**   |
|                   |                   |                              |
|                   |                   | NOAEL ≥ 1000 mg/kg/day       |
|                   |                   | (highest dose tested)        |
|                   |                   |                              |
|                   |                   | LOAEL \> 1000 mg/kg/day (not |
|                   |                   | established)                 |
+-------------------+-------------------+------------------------------+
| 870.5265 (§84-2)  | 93050017,         | **Negative**                 |
|                   | reformat of       |                              |
| *Salmonella       | 00088956          | There was no evidence of     |
| typhimurium*      |                   | induced mutant colonies over |
| reverse mutation  | **Unacceptable -  | background. Positive and     |
| test              | Guideline**       | solvent controls induced the |
|                   |                   | appropriate responses in the |
| Azad              | 0, 0.01, 0.05,    | strains in which they were   |
| ioxabicyclooctane | 0.23, 0.45, or    | tested; however, in the      |
| purity 50%        | 0.9 µL/plate      | absence of S9-mix, only      |
|                   |                   | strains TA1535 and TA1537    |
|                   |                   | were treated with a positive |
|                   |                   | control and in the presence  |
|                   |                   | of the S9-mix, only strains  |
|                   |                   | TA98, TA100, and TA1538 were |
|                   |                   | treated with a positive      |
|                   |                   | control.                     |
+-------------------+-------------------+------------------------------+
| 870.5265 (§84-2)  | 93050018,         | **Negative**                 |
|                   | reformat of       |                              |
| *Salmonella       | 00088974          | There was no evidence of     |
| typhimurium*      |                   | induced mutant colonies over |
| reverse mutation  | **Unacceptable -  | background. The positive     |
| test              | Guideline**       | controls induced the         |
|                   |                   | appropriate responses in the |
| Azad              | 0, 5, 10, 50,     | corresponding strains in the |
| ioxabicyclooctane | 100, or 250       | presence of S9-mix; however, |
| purity 50%        | µg/plate (initial | no positive control was used |
|                   | assay)            | in the absence of S9-mix.    |
|                   |                   |                              |
|                   | 0, 375, 500, 625, |                              |
|                   | or 750 µg/plate   |                              |
|                   | (second assay)    |                              |
+-------------------+-------------------+------------------------------+
| 870.5395 (§84-2)  | 41728601          | **Negative**                 |
|                   |                   |                              |
| Mammalian         | **Unacceptable -  | There was no significant     |
| erythrocyte       | Guideline**       | increase in the frequency of |
| micronucleus      |                   | micronucleated polychromatic |
| test - Mouse      | 0, 300, 600, or   | erythrocytes in bone marrow  |
|                   | 1200 mg/kg        | PCEs in either sex at any    |
| Azad              | (males)           | tested dose at either        |
| ioxabicyclooctane |                   | harvest time. No positive    |
| purity not        | 0, 600, 1200, or  | control was used in females. |
| reported          | 2400 mg/kg        | Positive and solvent control |
|                   | (females)         | values were acceptable.      |
+-------------------+-------------------+------------------------------+
| 870.5450 (§84-2)  | 240259303 and     | **Negative**                 |
|                   | 93050026,         |                              |
| Dominant Lethal - | reformat of       | There was no significant     |
| Rat               | 00088953          | difference between the       |
|                   |                   | control group and any        |
| Azad              | **Unacceptable -  | Nuosept 95 treated group     |
| ioxabicyclooctane | Guideline**       | with respect to              |
| purity 50%        |                   | fertilization rate, dead     |
|                   | 0, 125, 625, or   | implantations, living        |
|                   | 3125 pom          | implantations,               |
|                   |                   | preimplantation loss, or     |
|                   |                   | total implantations. The     |
|                   |                   | only statistically and       |
|                   |                   | biologically significant     |
|                   |                   | effect observed in the       |
|                   |                   | high-dose group was reduced  |
|                   |                   | water consumption and        |
|                   |                   | resultant reduced body       |
|                   |                   | weight. Because Nuosept 95   |
|                   |                   | was administered in the      |
|                   |                   | drinking water, this effect  |
|                   |                   | may be due to taste aversion |
|                   |                   | of the test substance.       |
+-------------------+-------------------+------------------------------+
| 870.5500 (§84-2)  | 93050019,         | **Positive**                 |
|                   | reformat of       |                              |
| Bacterial DNA     | 00088957          | There was evidence of        |
| Damage or repair  |                   | greater growth inhibition or |
| tests             | **Acceptable -    | cell killing in              |
| -*Escherichia     | Guideline**       | repair-defective strains     |
| coli* and         |                   | compared to repair competent |
| *Salmonella       | 0, 0.03, 0.3,     | strains. The negative        |
| typhimurium*      | 0.6, or 0.9 µL/mL | solvent and positive         |
|                   |                   | controls induced the         |
| Azad              |                   | appropriate responses in the |
| ioxabicyclooctane |                   | corresponding strains.       |
| purity 50%        |                   |                              |
+-------------------+-------------------+------------------------------+
| 870.5500 (§84-2)  | 93050020,         | **Positive**                 |
|                   | reformat of       |                              |
| Bacterial DNA     | 00088958          | There was evidence of        |
| Damage or repair  |                   | greater growth inhibition or |
| tests             | **Unacceptable -  | cell killing in              |
| -*Escherichia     | Guideline**       | repair-defective strains     |
| coli* and         |                   | compared to repair-competent |
| *Salmonella       | 0, 50, 100, 500,  | strains. The number of       |
| typhimurium*      | 1000, or 5000     | replicate plates and/or      |
|                   | µg/mL             | cultures was not given,      |
| Azad              |                   | individual plate counts were |
| ioxabicyclooctane |                   | not provided (just summary   |
| purity 50%        |                   | data), the S9-mix            |
|                   |                   | composition was not given,   |
|                   |                   | no information on the test   |
|                   |                   | material except the lot      |
|                   |                   | number was provided, and the |
|                   |                   | positive control for the *S. |
|                   |                   | typhimurium* strains without |
|                   |                   | S9-mix was not specified.    |
+-------------------+-------------------+------------------------------+
| 870.5550 (§84-2)  | 93050021,         | **Positive**                 |
|                   | reformat of       |                              |
| Unscheduled DNA   | 00088959          | There was evidence that      |
| Synthesis in      |                   | unscheduled DNA synthesis,   |
| Mammalian Cells   | **Unacceptable -  | as determined by radioactive |
| in Culture - Rat  | Guideline**       | tracer procedures \[nuclear  |
|                   |                   | silver grain counts\]. The   |
| Azad              | 0, 0.0156,        | controls induced the         |
| ioxabicyclooctane | 0.0316, 0.0625,   | appropriate responses.       |
| purity not        | 0.125, 0.25, 1.0, |                              |
| reported          | or 2.0 µL/mL      |                              |
+-------------------+-------------------+------------------------------+
| 870.5550 (§84-2)  | 42711001          | **Negative**                 |
|                   |                   |                              |
| Unscheduled DNA   | **Acceptable -    | There was no evidence that   |
| Synthesis in      | Guideline**       | unscheduled DNA synthesis,   |
| Mammalian Cells   |                   | as determined by radioactive |
| in Culture - Rat  | 0, 875, 1750, or  | tracer procedures \[nuclear  |
|                   | 3500 mg/kg        | silver grain counts\], was   |
| Azad              |                   | induced by Nuosept 95 as     |
| ioxabicyclooctane |                   | tested in this study. The    |
| purity not        |                   | positive and solvent         |
| reported          |                   | controls induced the         |
|                   |                   | appropriate responses.       |
+-------------------+-------------------+------------------------------+
| (§84-4)           | 93050022 reformat | **Positive**                 |
|                   | of 00088960       |                              |
| Other             |                   | Nuosept 95 was tested from   |
| Genotoxicity      | **Acceptable --   | nontoxic to toxic levels and |
|                   | Non-guideline**   | both positive and negative   |
| In Vitro Cell     |                   | controls were appropriate.   |
| Transofrmation    | 0, 0.0005, 0.001, | Treatment with Nuosept 95    |
| Assay in          | 0.002, or 0.004   | induced transformation of    |
| CH3/10T1/2 Cells  | µL/mL             | the CH3/10T1/2 cells. The    |
|                   |                   | transformation frequency was |
| Azad              |                   | low compared to the positive |
| ioxabicyclooctane |                   | control, there was no dose   |
| purity 50%        |                   | response, and no statistical |
|                   |                   | methods were employed to     |
|                   |                   | demonstrate significance;    |
|                   |                   | however, the spontaneous     |
|                   |                   | transformation frequency of  |
|                   |                   | the test system is virtually |
|                   |                   | zero, so any induction of    |
|                   |                   | transformed foci in an       |
|                   |                   | otherwise adequate           |
|                   |                   | experiment is regarded as a  |
|                   |                   | positive result.             |
+-------------------+-------------------+------------------------------+
| (§84-4)           | 93050023 reformat | **Positive**                 |
|                   | of 00088961       |                              |
| Other             |                   | No Type II or Type III foci  |
| Genotoxicity      | **Unacceptable -- | of transformed cells were    |
|                   | Non-guideline**   | observed in any treatment    |
| In Vitro Cell     |                   | group. Cytotoxicity was      |
| Transofrmation    | 0, 0.0003, 0.001, | evident at the high dose,    |
| Assay in          | or 0.003 µL/mL    | demonstrated by reduction of |
| CH3/10T1/2 Cells  |                   | relative cloning efficiency  |
|                   |                   | below 50%. Positive and      |
| Azad              |                   | solvent control values were  |
| ioxabicyclooctane |                   | appropriate.                 |
| purity not        |                   |                              |
| reported          |                   | This study is                |
|                   |                   | **unacceptable/              |
|                   |                   | non-guideline** because it   |
|                   |                   | cannot be ascertained        |
|                   |                   | whether T1597 could have     |
|                   |                   | been tested at a             |
|                   |                   | considerably higher dose and |
|                   |                   | because the duration of      |
|                   |                   | incubation of the cell       |
|                   |                   | cultures following treatment |
|                   |                   | was not precisely specified. |
+-------------------+-------------------+------------------------------+
| (§84-4)           | 93050024 reformat | **Negative**                 |
|                   | of 00088962       |                              |
| Other             |                   | R-1162 was tested from       |
| Genotoxicity      | **Acceptable --   | nontoxic to toxic levels.    |
|                   | Non-guideline**   | Positive and solvent control |
| In Vitro Cell     |                   | values were anomalous when   |
| Transofrmation    | 0, 0.0098,        | expressed as relative        |
| Assay in          | 0.0195, 0.039, or | cloning efficiency, probably |
| CH3/10T1/2 Cells  | 0.078 µL/mL       | due to low survival in the   |
|                   |                   | solvent control plates.      |
| Azad              |                   | Since survival in the test   |
| ioxabicyclooctane |                   | and positive control         |
| purity not        |                   | cultures was not reduced, a  |
| reported          |                   | toxic effect of the solvent  |
|                   |                   | is not indicated. No         |
|                   |                   | transformed foci were        |
|                   |                   | observed in the solvent      |
|                   |                   | controls, and the positive   |
|                   |                   | controls produced the        |
|                   |                   | expected number of           |
|                   |                   | transformed foci, so the     |
|                   |                   | assay remains valid.         |
|                   |                   | Treatment with R-1162 did    |
|                   |                   | not induce morphologic       |
|                   |                   | transformation of CH3/10T1/2 |
|                   |                   | cells.                       |
+-------------------+-------------------+------------------------------+
| (§84-4)           | 93050025 reformat | **Negative**                 |
|                   | of 00088963       |                              |
| Other             |                   | R-1143 was tested from       |
| Genotoxicity      | **Acceptable --   | nontoxic to toxic levels.    |
|                   | Non-guideline**   | Positive and solvent control |
| In Vitro Cell     |                   | values were appropriate.     |
| Transofrmation    | 0, 0.0006,        | Treatment with R-1143 did    |
| Assay in          | 0.0012, 0.0024,   | not induce morphologic       |
| CH3/10T1/2 Cells  | or 0.0048 µL/mL   | changes.                     |
|                   |                   |                              |
| Azad              |                   |                              |
| ioxabicyclooctane |                   |                              |
| purity not        |                   |                              |
| reported          |                   |                              |
+-------------------+-------------------+------------------------------+

^1^ - Daily doses estimated based on the analytical concentration of the
test article in water and the water intake data.

**9.0 REFERENCES**

41641601 (MRID #): Dilley, J.V. (Unknown). Acute oral toxicity of
Nuosept 95 in rats (Interim progress report). SRI International.

41641602 (MRID #): Hersham, R.J. (1984). Summary of results of a primary
eye irritation study. Bioresearch Incorporated.

41641603 (MRID #): Unwin, S.E. (1983). Primary dermal irritation test of
Nuosept® 95 in New Zealand White albino rabbits. Midwest Research
Institute.

41641605 (MRID #): Elliot, P.H., et al. (1985). Twenty-one Day Dermal
Toxicity Study in Rabbits with Nuosept 95/Nuosept C. Huntingdon Research
Centre.

41641606 reformat of 00109245 (MRID #): Sasmore, DP. (1980). Final
Report: Effect of Nuosept 95 in Rats 90-Day Toxicity Study - Contains
dose range study for Dominant Lethal Study (OPPTS 870.3100). SRI
International.

41671801 (MRID #): Liggett, M.P. and L.A. McRae. (1990). Acute dermal
toxicity to rabbits of Nuosept 95 (Final report). Huntingdon Research
Centre Ltd.

41699001 (MRID #): Smith, J.A., et al. (1988). A Study of the Effect of
Nuosept 95 on Pregnancy of the Rat. Huntingdon Research Centre.

41728601 (MRID #): O'Loughlin, K.G. (1990). Measurement of micronuclei
in bone marrow erythrocytes of Swiss-Webster mice following two
treatments with Nuosept 95. SRI International.

42650901 (MRID #): Cholakis, J.M. and H. Sprinz. (1983). Acute
inhalation toxicity of Nuosept 95 in Sprague-Dawley rats. Midwest
Research Institute.

42711001 (MRID #): Hamilton, C.M. (1983). Measurement of unscheduled DNA
synthesis in male Fischer-344 rat hepatocytes following *in vivo*
treatment with Nuosept 95. SRI International.

93050017 reformat of 00088956 (MRID #): Anonymous (1979).
*Salmonella*/mammalian-microsome plate incorporation mutagenesis assay.
An Evaluation of Carcinogenic Potential of NUOSEPT® 95 Employing the
C3H/10T½ Cell Transformation Assay. EG&G Mason Research Institute.

93050018 reformat of 00088974 (MRID #): Van Goethen, D. (1979).
Mutagenicity studies on Nuosept 95 *Salmonella*/microsome test. Midwest
Research Institute.

93050019 reformat of 00088957 (MRID #): Anonymous (1979). Bacterial DNA
damage/repair suspension assay. EG&G Mason Research Institute.

93050020 reformat of 00088958 (MRID #): Anonymous (1979). Bacterial DNA
repair assay. Midwest Research Institute.

93050021 reformat of 00088959 (MRID #): Anonymous (1980). Evaluation of
Nuosept ® 95 in the primary rat hepatocyte unscheduled DNA synthesis
assay. Litton Bionetics, Inc.

93050022 reformat of 00088960 (MRID #): Thilagar, A., et al. (1979). An
Evaluation of Carcinogenic Potential of NUOSEPT® 95 Employing the
C3H/10T½ Cell Transformation Assay. EG&G Mason Research Institute.

93050023 reformat of 00088961 (MRID #): Schechtman, M. (1980). Activity
of T1597 in an in vitro Mammalian Cell transformation Assay in the
Absence of Exogenous Metabolic Activation. Microbiological Associates.

93050024 reformat of 00088962 (MRID #): Thilagar, A., et al. (1980). An
Evaluation of Carcinogenic Potential of R-1162 (NUOSEPT® 95) Employing
the C3H/10T½ Cell Transformation System. EG&G Mason Research Institute.

93050025 reformat of 00088963 (MRID #): Thilagar, A. (1980). An
Evaluation of Carcinogenic Potential of R-1143 (NUOSEPT® 95) Employing
the C3H/10T½ Cell Transformation System. EG&G Mason Research Institute.

93050026 reformat of 00088953 (MRID #): Anonymous (1979). Dominant
lethal study of Nuosept 95. SRI International.

McMahon, Timothy, et al. (2005). Azadioxabicyclooctane - Report of the
Antimicrobial Division Toxicology Endpoint Selection Committee.