Mammalian Toxicology and Human Health Worker Exposure Safety Assessment

Supporting Study Mammalian Safety Assessment Data

The Mammalian Toxicology and Human Health Worker Exposure Safety Assessment are based on the study data for the representative EXP 1313100 (Table 1). One additional study, Repeated

Dose 28- Day Dermal Toxicity Study, is in progress and the final report will be submitted to EPA when received. In addition, EXP1313100 is an alkaryl sulfonate and as such fits into the US EPA High Production Volume (HPV) alkaryl sulfonate category (Table 2). All studies

conducted on EXP1313100 as well as the publicly available study summaries and EPA decision document for the HPV category are included as attachments to this PMN applicatio n (Attachments 034-036).

For HPV evaluation the p etroleum additive a lkaryl s ulfonate category was defined as complex mixtures designated as Class 2 substances for TSCA Inventory Reporting. Class 2 substances, in general, have a comple x and variable composition depending on their source or methods of derivation. The sulfonated alkylbenzenes are manufactured by the sulfonation of either synthetic alkylbenzene substrates or naturally occurring alkylaromatic -rich fractions of heavy lubric ating oil base stocks derived from petroleum streams. The alkyl substituent group may vary in number (e.g., mono- or dialkyl), position (e.g., predominantly meta or para to the sulfonic acid position), chain length (e.g., C14 to C30) or in the degree of branching. Branched and linear alkyl groups of 20 or more carbons are used to enhance oil solubility. The substances in this category include

sulfonic acids as well as their calcium, sodium, barium and magnesium salts.

This class 2 substance thus fits within the definition of the pe troleum additive a lkaryl sulfonate category and supports the use of this category’s testing data.

1Table 1 . Mammalian Safety Assessment Supporting Studies Conducted on EXP1313100

Title Summary (NOAELs in mg/kg bw/day) Attachment

#

Assessment of Acute Oral Toxicity with EXP1313100 in the Rat (Acute Toxic Class Method). Unpublished Project 505697. WIL Research Europe B.V. Hertogenbosch, The Netherlands. February 6, 2015. Oral LD 50 : >2,000 mg/kg bw 011

Assessment of Acute Dermal Toxicity with EXP1313100 in the Rat. Unpublished Project 505698. WIL Research Europe B.V. Hertogenbosch, The Netherlands. February 6, 2015. Dermal LD 50 : >2,000 mg/kg bw 012

Acute Eye Irritation/Corrosion Study with EXP1313100 in the Rabbit. Unpublished Project 50 7 908 . WIL Research Europe B.V. Hertogenbosch, The Netherlands. February 12, 2015. EXP1313100 as a 50% (w/w) dilution in propylene glycol was

n on

– irritating. Mild

i rritation (score of 1,

non

- positive) resolved

within 4 8 hours in all

animals 013

Screening fo r the Eye Irritancy Potential of EXP1313100 using the Bovine Corneal Opacity and Permeability Test (BCOP Test). Unpublished Project 505701. WIL Research Europe B.V. Hertogenbosch, The Netherlands. July 22, 2014. Mild to moderate ocular irritation, no pred iction

on classification 014/015

Screening for the Eye Irritancy Potential of EXP1313100 10% (W/V) in Propylene Glycol using the Bovine Corneal Opacity and Permeability Test (BCOP Test). Unpublished Project 50 7 656. WIL Research Europe B.V. Hertogenbosch, The Netherlands. January 20, 2015. Mild to moderate ocular irritation, no prediction

on classification 016

In Vitro Skin Irritation Test with EXP1313100 using a Human Skin Model. Unpublished Project 505700. WIL Research Europe B.V. Hertogenbosch, The Netherlands. July 9, 2014. Non

- irritating 017

14

- Day Range Finding Study with EXP1313100 by Daily Gavage or Dermal Exposure in the Rat. Unpublished Project 505704. WIL Research Europe B.V. Hertogenbosch, The Netherlands. January 30,

2015. Oral NOAEL = 1,000 mg/kg bw/day

Dermal NOAEL = 1,000 mg/kg bw/day with mild

local irritation 018

Repeated Dose 28

- Day Oral Toxicity Study with EXP1313100 by Daily Gavage in the Rat Followed by a 14

- Day Recovery Period. Unpublished Project 50570 5 . WIL R esearch Europe B.V. Hertogenbosch, The Netherlands. February 23 , 2015

. NOAEL = 300 mg/kg bw/day 019

Acute Eye Irritation/Corrosion Study with EXP1313100 in the Rabbit. Unpublished Project 505702. WIL Research Europe B.V. Hertogenbosch, The Netherlands. Draft Report EXP1313100 as 100%

brown solid was unable

to be tested because material was expelled from the eye once eyelid

was released 020

Evaluation of the Mutagenic Activity of EXP1313100 in the Salmonella Typhimurium Reverse Mutation Assay and the Escherichia Coli Reverse Mutation Assay. Unpublished Project 505689. WIL Research Europe B.V. Hertogenbosch, The Netherlands . July 25, 2014

. Negative with and

without metabolic

activation 021/022

Evaluation of the Ability of EXP1313100 to Induce Chromosome Aberrations in Cultured Peripheral Human Lymphocytes. Unpublished Project 505690. WIL Research Europe B.V. Hertogenbosch, The

Netherlands

. October 14 , 2014

. Negative with and

without metabolic

activation 023/024

Micronucleus Test in Bone Marrow Cells of the Rat with EXP1313100

. Unpublished Project 505691. WIL Research Europe B.V. Hertogenbosch, The Netherlands

. November 3, 2014. Negative 025

NOAEL = no observable adverse effect level

2Table 2 . Mammalian Safety Assessment for EPA HPV Alkaryl Sulfonate Category

Title Summary (NOAELs in mg/kg bw/day) Attachment

#

Acute Oral Toxicity Oral LD 50 : >2,000 mg/kg

bw 034 Table 3

Acute Dermal Toxicity Dermal LD 50 : >2,000 mg/kg bw 034 Table 3

Acute Inhalation Toxicity Inhalation LC 50 : >1.9 mg/L (maximum attainable concentration) 034 Table 3

28

- Day Oral Repeated Dose Study in Rats (OECD 407) NOAEL = 500 mg/kg bw/day

LOAEL = 1000 mg/kg bw/day (decreased serum cholesterol) 034 Table 3

28

- Day Dermal Repeated Dose Study in Rats (OECD 410) NOAEL (male/female) =

1000 mg/kg bw/day (HDT) 034 Table 3

28

- Day Inhalation Repeated Dose Study in Rats ( OECD 412) NOAEL = 49.5 mg/m 3 035 Table 9

Oral One

- Generation Reproduction Study in Rats (OECD 415) NOAEL = 500 mg/kg bw/day 034 Table 3

Bacterial Reverse Mutation Assay (OECD 471/472) Negative with and without

metabolic activation 034 Table 3

Mammalian Gene Mutation Assay (OECD 476) Negative with and without

metabolic activation 034 Table 3

In Vitro Chromosome Aberration Assay (OECD 473) Negative with and without

metabolic activation 034 Table 3

In Vivo Chromosome Aberration Assay (OECD 474) Negative 034 Table 3
NOAEL = no observable adverse effect level; HDT = highest dose level tested

Safety Data Sheets ( SDS)

A SDS shee t has been provided for EXP1 313100

• Attachment 006

Human Health EXP 1313100 Potential Exposure and Risk

The available toxicological testing with EXP1 313100 indicate that it is of low acute oral and dermal toxicity (LD 50 s >2,000 mg/kg bw) and it is non-irritating to the skin and eyes. The results from the acute toxicity testing available for EXP1313100 are in agreement with the results from the testing of the alkaryl sulfonate category.

Sub-acute and subchronic oral toxicity testing (14 - and 28-day studies , respectively) are available for EXP1313100. The testing indicates an oral NOAEL of 300 mg/kg bw/d ay from the 28-day study, with effects at the LOAEL of 1,000 mg/kg bw/day. This is in accordance with the

28- day repeated dose data available for the alkaryl sulfonate category, which indicates a NOAEL of 500 mg/kg bw/day with any differences due to dose spacing in the available studies.

3EXP1 313100 has been tested in in vitro and in vivo genotoxicity studies including a bacterial reverse mutation assay, a n in vitro chro mosome aberrations study and an in vivo micronucleus assay. Based on the results of the genotoxicity testing, EXP1313100 is not a genotoxic concern. This is in accordance with the genotoxicity data available for the alkaryl sulfonates category.

No reproductive, developmental or teratogenic data are available on EXP1313100. The data

ava ilable for the alkaryl sulfonate category indicate a NOAEL of 500 mg/kg bw/day for reproductive toxicity, maternal toxicity and developmental toxicity, the highest dose tested.



### Acute Toxicity Testing

Acute Oral Toxicity with EXP1313100: Assessment of Acute Oral Toxicity with EXP1313100
in the Rat (Acute Toxic Class Method). Unpublished Project 505697. WIL Research Europe B.V.
Hertogenbosch, The Netherlands. February 6, 2015.

The acute oral toxicity of EXP1313100 was investigated in this study, which was conducted

under GLP and in accordance with OECD Guideline 423 and EPA Guideline OPPTS 870.1100. In this study, EXP1313100 was administered to female Wistar rats (3/dose) at a dose level of 2,000 mg/kg bw by gavage. No mortality was observed, and thr ee additional female animals were dosed at 2,000 mg/kg bw. Mortality, clinical observations, and body weight changes were

evaluated over a 14

- day observation period. All animals were subjected to a gross necropsy.

There were no deaths, body weight chang es or gross necropsy findings. Clinical effects included lethargy, hunched posture and/or piloerection for all animals on Days 1 and/or 2.

Under the conditions of this study, the acute oral LD 50 was > 2,000 mg/kg bw.

Acute Dermal Toxicity with EXP1313100: Assessment of Acute Dermal Toxicity with
EXP1313100 in the Rat. Unpublished Project 505698. WIL Research Europe B.V.
Hertogenbosch, The Netherlands. February 6, 2015.

The acute dermal toxicity of EXP1313100 was inves tigated in this study, which was conducted

under GLP and in accordance with OECD Guideline 402 and EPA Guideline OPPTS 870.1200. In this study, EXP1 313100 was administered to Wistar rats (5/sex/dose) at a dose level of 2,000 mg/kg bw, dermally over a 24 h our period under a semi

- occlusive dressing. Mortality, dermal findings and body weight changes were evaluated over a 14

- day observation period. All animals were subjected to a gross necropsy.

There were no deaths, remarkable body weight changes or gross necropsy findings. Dermal findings consisted of erythema (general or maculate), fissuring, scales and/or scabs were seen on

the treated skin

- area and/or left flank of all animals during the observation period. Additionally,

all females showed necrosis o f the treated skin

- area and/or left flank between Days 4 and 8. One of these females had a wound on the left flank on Days 7 and 8 and scar formation on the left flank on Days 14 and 15.

Under the conditions of this study, the acute dermal LD 50 was > 2,000 mg/kg bw.

4In Vivo Eye Irritation with EXP 1313100 : Acute Eye Irritation/Corrosion Study with EXP1313100 in the Rabbit. Unpublished Project 50 7 908. WIL Research Europe B.V. Hertogenbosch, The Netherlands. February 12, 2015

.

The purpose of this acute eye irritation/corrosion study was to assess the possible irritation or corrosion potential when a single dose of the test substance was placed in the conjunctival sac of the rabbit eye. T his study was conducted under GLP and according to OECD Guide line 405 and

OPPTS Guideline 870.2400. This study was conducted, because classification based on the in

vitro eye irritation studies was not possible. In this study, EXP1313100 was prepared as a 50% (w/w) dilution in propylene glycol

. An i nitial in vivo eye irritation study was conducted on a neat sample of EXP1313100 (Project 505702) . The neat substance was applied to the eye of one

rabbit. However, the test substance fell out of the eye immediately after releasing the eyelids

after instillation. There fore, application of the test substance was considered to have resulted in an insufficiently long exposure period to allow a meaningful interpretation of the eye irritation

hazard

. To overcome the instillation issue, a follow up study was performed (Proje ct 505908) using EXP1313100 diluted in a vehicle. Propylene glycol was selected because EXP1313100 has

been shown to be stable in this vehicle, a 50% (w/w) dilution was selected as this was the maximum concentrations that formed a homogenous formulation

.

EXP 1313100 was initially applied ocularly to one rabbit (unwashed). No severe ocular damage

was observed in this animal. Two additional rabbits received single unwashed exposures of

EXP1 3131100 . Each 0.1 mL dose of the test substance was instilled into the lower conjunctival sac of the right eye. The eyelid was held closed for approximately 1 second and released. The left eye of each animal was manipulated in an identical manner to simulate t he dosing of the right eye and served as a contralateral control. In all animals, conjunctival irritation was graded as 1 (not positive) and subsided by 48 hours

.

Under the conditions of this study, EXP 1313100 as a 50% (w/w) dilution in propylene glycol is

considered non

- irritating to the eyes.

In Vitro Eye Irritation with EXP1 313100 : Screening for the Eye Irritancy Potential of EXP1313100 using the Bovine Corneal Opacity and Permeability Test (BCOP Test). Unpublished Study No. Project 505701. WIL Re search Europe B.V. Hertogenbosch, The Netherlands. July 22, 2014.

EXP1 13100 was tested for potential eye irritation in the in vitro Bovine Corneal Opacity and Permeability test (BCOP test) in accordance with OECD Guideline 437 under Good Laboratory Practice (GLP). The possible ocular irritancy of EXP 1313100 to isolate d corneas from bovine

eyes was tested through topical application of undiluted material for 10 ± 1 minute s. The test

substa nce was added as is (sticky brown solid) via a gauze patch of approximately 2 cm 2 directly on top of the cornea (approximately 400 mg). The negative control (physiological saline) responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in

vitro irritancy score of t he positive control (10% (w/v) benzalkonium chloride) was 126 and was

5within the historical positive contro l data range. The tes t conditions were adequate and the test system functioned properly. The corneas treated with EXP1 313100 showed opacity values ranging from 3 to 5 and permeability values ranging from 0.01 9 to 0.02 9

. The corneas were

translucent with spots after the 10 minutes of treatment with EXP1 313100

. No pH effect of the

test substance was observe d on the rinsing medium. T he in vitro irritancy scores ranged from

- 3.3 to 5.3 after 10 minutes of treatment with EXP 1313100

. EXP 1313100 induced ocular irritation through both endpoints in the category mild to moderate, resulting in a mean in vitro irritancy score (IVIS) of 4.3 after 10 minutes of treatment. Since EXP1313100 induced an IVIS > 3 ≤ 55.1, no prediction on the classification can be ma de.

In Vitro Eye Irritation with EXP1 313100: Screening for the Eye Irritancy Potential of EXP1313100 10% (W/V) in Propylene Glycol using the Bovine Corneal Opacity and Permeability Test (BCOP Test). Unpublished Study No. Project 50 7 656. WIL Research Europe B.V. Hertogenbosch, The Netherlands. January 20, 2015.

A 10% solution of EXP1 13100 in propylene glycol (W/V) was tested for potential eye irritation

in the in vitro Bovine Corneal Opacity and Permeability test (BCOP test) in accordanc e with OECD Guideline 437 under Good Laboratory Practice (GLP). The possible ocular irritancy of

EXP 1313100 to isolated corneas from bovine eyes was tested through topical application of

undiluted material for 10 ± 1 minute s. The test substance was appli ed as a 10% (w/v) suspension in propylene glycol directly on top of the corneas. The mean in vitro irritancy score of the solvent control (p ropylene glycol) was 1.0 indicating that the solvent control did not induce irritancy on the corneas. The negative control (physiological saline) responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of t he positive control (10% (w/v) benzalkonium chloride) was 1 11 and was within the historical positive control data range. The tes t conditions were adequate and the test system functioned properly. The corneas treated with EXP1 313100 showed opaci ty values ranging from 3 to 5 and permeability values ranging from 0.0 02 to 0.014

. The corneas were clear after the 10 minutes of

treatment with EXP1 313100

. No pH effect of the test substance was observe d on the rinsing

medium. T he in vitro irritancy sc ores ranged from

- 3.2 to 5. 0 after 10 minutes of treatment with

EXP 1313100

. EXP 1313100 induced ocular irritation through only the endpoint opacity, resulting in an in vitro irritancy score (IVIS) of 4. 1 after 10 minutes of treatment. Since

EXP1313100 induced an IVIS > 3 ≤ 55.1, no prediction on the classification can be made.

In Vitro Skin Irritation with EXP 1313100 : In Vitro Skin Irritation Test with EXP1313100 using a Human Skin Model. Unpublished Study No. Project 505700. WIL Research Europe B.V. Hertogenbosch, The Netherlands. July 9, 2014.

This report describes the ability of EXP 1313100 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN

- SM TM )) under GLP and in

acc ordance with OECD Guideline 439

. The possible skin irritation potential of EXP 1313100 was tested through topical application for 15 minutes. Skin tissue was moistened with 5 μl of

Milli

- Q water and 10.2 to 10.9 mg of EXP1313100 was applied directly on top of th e skin tissue

for 15 minutes. After a 4 2

- hour post

- incubation period, determination of the cytotoxic (irritancy)

6effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute

mean OD 570 (optical density at 570 nm) of the negative control tissues was within the laboratory

historical control data range. The standard deviation value of the percentage viability of three

tissues treated identically was less than 9%, indicating that the test system functioned properly.

Skin irritation is expressed as the remaining cell viability after e xposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with EXP1313100 compared to the negative control tissues was 120%. Since the mean relative tissue viability for

EXP1313100 was above 50% after 15 minute s treatment

, EXP1313100 is considered to be non

irritant.

Under the conditions of this study, EXP1 313100 is considered to be non

- irritating to the skin under the conditions of this study.



### Repeated Dose Oral Toxicity Testing

14

- Day Oral and Dermal Toxicity Study with EXP 1313100 in Rats: 14

- Day Range Finding Study with EXP1313100 by Daily Gavage or Dermal Exposure in the Rat. Unpublished Project 505704. WIL Research Europe B.V. Hertogenbosch, The Netherlands. January 30, 2015

This study was conduct ed to assess the toxic potential of the test substance when administered to rats by daily oral gavage or dermal exposure for a period of 14 days . The study was conducted under GLP and based around OECD Guideline 407 and 410. In this study, EXP1313100 was administered in propylene glycol at dose levels of 0 (vehicle control), 500 and 1,000 mg/kg bw/day to SPF

- bred Wistar rats for a period of 14 days

. For each route of exposure, one control group and two treated groups were tested, each consisting of 3 mal es. The dose volume for oral gavage was 5 mL/kg. For dermal exposure, t he test substance formulation was applied onto a surgical gauze patch with an area of approximately 10% of the total body surface (i.e. approx. 40 cm2) and the animals were exposed for 6 hours per day / 7 days a week

. The skin was then

cleaned of residual test substance using water

. Formulation analyses were not conducted in this study but the results from the 28

- day oral study were acceptable (Unpubli shed project number

50570 5 ). All animals were observed twice daily for mortality and moribundity. Clinical

examinations daily at 0

- 15 minutes, 1 hour and 3 hours after oral dosing and prior to and immediately after dermal exposure were performed at least once daily and detailed clinical examinations were performed immediately after dosing throughout the study. Body weights and food consumption were recorded weekly. Functional observational battery (FOB) and locomotor activity data were recorded for all animals during study week 4

. Clinical pathology parameters were not included in this study. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. No histopathology was performed in this study.

There were no test

- substance related deaths from oral or dermal exposure . No toxicologically relevant clinical signs were reported from oral exposure. Dermal exposure resulted in slight to moderate erythema, scabs and scales were reported in controls and treated groups, but the

7incidence and severity in the treated groups was higher than that of controls. Food consumption

remained similar to control level in both the dermal and oral treatment groups. Oral exposure resulted in a slight decrease in body w eight and body weight gain in the treatment groups. No effect on body weight or body weight gain was reported from dermal exposure. All animals in the dermal exposure treatment groups at 500 and 1

, 000 mg/kg /bw day showed scales and/or scab

formation of the treated skin area. No other macroscopic lesions were reported to be treatment related in the oral or dermal treatment groups and organ weights were considered to be unaffected by oral or dermal exposures.

O ra l exposure did not result in toxicologically relevant findings up to a dose of 1

, 000 mg/kg bw/day

. Dermal exposure resulted in generally mild local effects on the treated skin area

due to irritation. No evidence of systemic toxicity was noted after dermal exposure

.

28

- Day Oral Toxicity Study with EXP 1313100 in Rats: Repeated Dose 28

- Day Oral Toxicity Study with EXP1313100 by Daily Gavage in the Rat Followed by a 14

- Day Recovery Period. Unpublished Project 50570 5 . WIL Research Europe B.V. Hertogen bosch, The Netherlands. February 23 , 2015

This study was conducted to assess the toxic potential of the test substance when administered to rats by daily oral gavage for a period of 28 days, followed by a 14

- day recovery period. The study was conducted under GLP and according to OECD Guideline 407 and EPA Guideline

870.3050. In this study, EXP 1313100 was administered in propylene glycol at dose levels of 0 (vehicle control), 100, 300 and 1,000 mg/kg bw/day to SPF

- bred Wistar ra ts (5/sex/dose) by gavage for a period of 28 days. An extra 5 animals per sex in the control and high dose group

were maintained for 14

- days after the end of the test substance administration (recovery group).

The dose volume was 5 mL/kg for all groups. Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were

stable over at least 6 hours. All animals were observed twice daily for mortality and moribundity. Clinical examin ations were performed at least once daily and detailed clinical

examinations were performed immediately after dosing throughout the study. Body weights and food consumption were recorded weekly. Functional observational battery (FOB) and locomotor

activi ty data were recorded for all animals during study week 4

. Clinical pathology parameters (hematology, coagulation, and serum chemistry

- urinalysis was not conducted ) were analyzed for all rats on the day of the scheduled necropsy (study day 28). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues (including gross lesions) were examined microscopically from all animals in the control

and 1

, 000 mg/kg/day groups, and gross lesions w ere examined microscopically from all animals in the 100 and 300 mg/kg/day groups.

There were no test

- substance related deaths. No toxicologically relevant clinical signs were reported, intermittent salivation reported in the high dose group was consid ered to be a physiological response to the taste of the test substance. Food consumption remained similar to

control level. No treatment

- related effects were reported for FOB evaluations.

Body weight gain was lower (statistically significant on most occ asions) during the treatment period and in the recovery period for males in the 1,000 mg/kg bw/day dose group when

8compared with the vehicle control group. Mean body weight was also statistically significantly

lower for both sexes at commencement of the r ecovery phase, but mean weight gain over the recovery period compared to Day 1 of the recovery period was similar to control levels for both

sexes.

One male in the 1

, 000 mg/kg bw/day treatment group showed a slight multifocal necrosis of the liver, whic h was considered to be related to treatment. Treatment

- related changes in clinical biochemistry parameters at 1

, 000 mg/kg bw/day that would support hepatocellular damage included higher alanine and aspartate aminotransferase activity in both sexes (alanine aminotransferase activity was increased with a factor of approximately 12 and 7 for males and females, respectively), and higher alkaline phosphatase activity in males. Other clinical biochemistry changes at the end of treatment that were conside red to be related to treatment at

1

, 000 mg/kg bw/day consisted of a lower total protein, albumin, cholesterol and calcium level and higher total bilirubin and bile acid level in both sexes, and lower creatinine level in males.

At the end of the recovery p eriod, multifocal necrosis of the liver was absent and only total protein level remained lower in males at 1

, 000 mg/kg bw/day

. Given the magnitude of change of primarily alanine aminotransferase activity at 1

, 000 mg/kg bw/day , in combination with the

obse rved liver necrosis in a single male and other clinical biochemistry changes that point to a disturbance in liver function, it is considered that an adverse effect on liver integrity occurred at

this dose.

The NOAEL was considered to be 30 0 mg/kg bw/day based on liver

- related effects at the LOAEL of 1,000 mg/kg bw/day.



### Genotoxicity Testing

Bacterial Reverse Mutation Assay Evaluation of the Mutagenic Activity of EXP1313100 in the Salmonella Typhimurium Reverse Mutation Assay and the Escherichia Coli Reverse Mutation Assay. Unpublished Project 505689. WIL Research Europe B.V. Hertogenbosch, The Netherlands. July 25, 2014

This study was conducted to evaluate the in vitro mutagenic potential in bacterial cells and was conducted under GLP and according to OECD guideline 471. In this study, EXP 1313100 was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester str ain WP2 uvr A in the presence

and absence of Aroclor

- induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity

- mutation assay, was used to

establish the dose

- range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was the s olvent

. The test article formed a clear brown solution in

DMSO. In the init ial toxicity

- mutation assay, EXP1313100 was tested at 1. 7 , 5. 4 , 17, 52, 164

, 5 12 , 1

, 600 and 5

, 000 μg/ plate in triplicate in the presence and absence of S9. The highest

concentrat ion used in the subsequent mutation assay was 5 ,000 μg/plate. Precipitation of EXP1313100 on the plates was not observed at the start or at the end of the incubation period in

9any tester strain in the first or second phase

. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed in the first or second phase

.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation in the first or second phase.

Under the conditions of this study, test article EXP1 313100 was concluded to be negative in the Bacterial Reverse Mutation Assay.

In Vitro Chromosome Aberration Assay : Evaluation of the Ability of EXP1313100 to Induce Chromosome Aberrations in Cultured Peripheral Human Lymphocytes. Unpublished Project 505690. WIL Research Europe B.V. Hertogenbosch, The Netherlands. October 14, 2014

This study was conducted to evaluate the ability of EXP1313100 to induce chromosome

abe rrations in cultured human peripheral human lymphocytes (HPBL) and was conducted under GLP and in accordance with OECD Guideline 473. The solvent in the study was dimethyl sulfoxide (DMSO).

In the first cytogenetic assay, EXP1313100 was tested up to 51 2 μg/mL for 3 hours with a 24 hour fixation time with and without S9 activation. EXP1313100 precipitated at 512 μg/mL

. In the second cytogenetic assay, EXP1313100 was tested up to 125 μg/mL for 24 hours with a 24 hour fixation time in and up to 100 μg/mL for 48 hours with a 48 hours fixation time in the

absence of S9. Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was

within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells

with chromosome aberrations, indicating that the test conditions were adeq uate and that the

metabolic activation system (S9

- mix) functioned properly.

EXP1313100 did not induce a statistically significant or biologically relevant increase in the

number of cells with chromosome aberrations in the absence and presence of S9

- mix, in either of

the two independently performed experiments. No biologically relevant effects of EXP1313100 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed in both the absence and presence of S9

- mix. Therefore, it can be concluded that EXP1313100 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that EXP1313100 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

In Vivo Micronucleus Assay: Micronucleus Test in Bone Marrow Cells of the Rat with EXP1313100. Unpublished Project 505691. WIL Research Europe B.V. Hertogenbosch, The Netherlands. November 3, 2014.

10In this study, EXP 1313100 was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone

marrow under GLP and in accordance with OECD Guideline 474

. Propylene glycol was selected as the vehicle. Test and/or contr ol substance formulations were administered at a dose

volume of 5 mL/kg by oral gavage. The test species was Wistar rats.

The concentrations analyz ed in the formulations and the stability of the formulations were acceptable.

In the dose range finding st udy 3 males and 3 females were dosed via oral gavage with 2

, 000

mg /kg bw

. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no substantial differences in toxicity between sexes

, only males were used in

the main study.

In the main study male animals were dosed by oral gavage with vehicle or with 500, 1,000 or

2 ,000 mg/kg bw

. A positive control group was dosed once by oral gavage with cyclophosphamide (CP) at 20 mg/kw bw

. In total 6 treatment groups were used, each consisting

of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with EXP1313100 or control animals receiving vehicle or cyclophosphamide.

Bone marrow of the groups t reated with EXP1313100 was sampled 24 or 48 (highest dose only)

hours after dosing. Bone marrow of the negative and positive control groups was harvested 24

and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated po lychromatic erythrocytes was observed

in the bone marrow of animals treated with EXP1313100 compared to the vehicle treated

animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative

control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were

met.

The groups that were treated with EXP1313100 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that

was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic

to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that EXP1313100 is n ot clastogenic or aneugenic in the bone marrow

micronucleus test when sampled at 24 and 48 hours post dosing of male rats up to a dose of

2

, 000 mg/kg bw the recommended dose in accordance with current regulatory guidelines under the experimental conditions described in this report.

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