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 EXP1313100 (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 application 

(Attachments 034-036). 

For HPV evaluation the petroleum additive alkaryl sulfonate category was defined as complex 

mixtures designated as Class 2 substances for TSCA Inventory Reporting. Class 2 substances, in 

general, have a complex 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 lubricating 

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 petroleum additive alkaryl sulfonate category and supports the use of this 

category’s testing data.

1 
Table 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 Unpublished Project 505697.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 LD50: >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Unpublished Project 505698.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 LD50: >2,000 

mg/kg bw 012

Acute Eye Irritation/Corrosion Study with EXP1313100 in the Rabbit. 

Unpublished Project 507908. WIL Research Europe B.V. 

Hertogenbosch, The Netherlands. February 12, 2015.

EXP1313100 as a 50% 

(w/w) dilution in 

propylene glycol was 

non –irritating. Mild 

irritation (score of 1, 

non-positive) resolved 

within 48 hours in all 

animals

013

Screening for 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 prediction 

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 507656. 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 505705. WIL Research 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

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

Title Summary (NOAELs in 

mg/kg bw/day)

Attachment 

#

Acute Oral Toxicity Oral LD50: >2,000 mg/kg 

bw 034 Table 3

Acute Dermal Toxicity Dermal LD50: >2,000 

mg/kg bw 034 Table 3

Acute Inhalation Toxicity

Inhalation LC50: >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/m3 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 sheet has been provided for EXP1313100 

• Attachment 006

Human Health EXP1313100 Potential Exposure and Risk 

 

The available toxicological testing with EXP1313100 indicate that it is of low acute oral and 

dermal toxicity (LD50s >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/day 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. 

3 
EXP1313100 has been tested in in vitro and in vivo genotoxicity studies including a bacterial 

reverse mutation assay, an in vitro chromosome 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 

available 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 three 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 changes 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 LD50 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 investigated in this study, which was conducted 

under GLP and in accordance with OECD Guideline 402 and EPA Guideline OPPTS 870.1200. 

In this study, EXP1313100 was administered to Wistar rats (5/sex/dose) at a dose level of 2,000 

mg/kg bw, dermally over a 24 hour 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 of 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 LD50 was > 2,000 mg/kg bw. 

4
In Vivo Eye Irritation with EXP1313100: Acute Eye Irritation/Corrosion Study with 

EXP1313100 in the Rabbit. Unpublished Project 507908. 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. This study was conducted under GLP and according to OECD Guideline 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 initial 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. Therefore, 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 (Project 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. 

EXP1313100 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 

EXP13131100. 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 the 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, EXP1313100 as a 50% (w/w) dilution in propylene glycol is 

considered non-irritating to the eyes. 

In Vitro Eye Irritation with EXP1313100: Screening for the Eye Irritancy Potential of 

EXP1313100 using the Bovine Corneal Opacity and Permeability Test (BCOP Test). 

Unpublished Study No. Project 505701. WIL Research Europe B.V. Hertogenbosch, The 

Netherlands. July 22, 2014.

EXP113100 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 EXP1313100 to isolated corneas from bovine 

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

substance was added as is (sticky brown solid) via a gauze patch of approximately 2 cm2 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 the positive control (10% (w/v) benzalkonium chloride) was 126 and was 

5
within the historical positive control data range. The test conditions were adequate and the test 

system functioned properly. The corneas treated with EXP1313100 showed opacity values 

ranging from 3 to 5 and permeability values ranging from 0.019 to 0.029. The corneas were 

translucent with spots after the 10 minutes of treatment with EXP1313100. No pH effect of the 

test substance was observed on the rinsing medium. The in vitro irritancy scores ranged from -

3.3 to 5.3 after 10 minutes of treatment with EXP1313100. EXP1313100 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 made.

In Vitro Eye Irritation with EXP1313100: 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 507656. WIL Research Europe 

B.V. Hertogenbosch, The Netherlands. January 20, 2015.

A 10% solution of EXP113100 in propylene glycol (W/V) 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 

EXP1313100 to isolated corneas from bovine eyes was tested through topical application of 

undiluted material for 10 ± 1 minutes. The test substance was applied 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 (propylene 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 the 

positive control (10% (w/v) benzalkonium chloride) was 111and was within the historical 

positive control data range. The test conditions were adequate and the test system functioned 

properly. The corneas treated with EXP1313100 showed opacity values ranging from 3 to 5 and 

permeability values ranging from 0.002 to 0.014. The corneas were clear after the 10 minutes of 

treatment with EXP1313100. No pH effect of the test substance was observed on the rinsing 

medium. The in vitro irritancy scores ranged from -3.2 to 5.0 after 10 minutes of treatment with 

EXP1313100. EXP1313100 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 EXP1313100: 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 EXP1313100 to induce skin irritation on a human three 

dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)) under GLP and in 

accordance with OECD Guideline 439. The possible skin irritation potential of EXP1313100

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 the skin tissue 

for 15 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) 

6
effect 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 OD570 (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 exposure 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 minutes treatment, EXP1313100 is considered to be nonirritant.

Under the conditions of this study, EXP1313100 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 EXP1313100 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 conducted 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 males. The dose volume for oral 

gavage was 5 mL/kg. For dermal exposure, the 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 (Unpublished project number 

505705). 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 

7
incidence 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 weight 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. 

Oral 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 EXP1313100 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 505705. WIL Research Europe B.V. Hertogenbosch, 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, EXP1313100 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 rats (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 examinations 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 

(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 were 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 considered 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 occasions) during the treatment 

period and in the recovery period for males in the 1,000 mg/kg bw/day dose group when 

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

lower for both sexes at commencement of the recovery 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, which 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 considered 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 period, 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 

observed 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 300 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, EXP1313100 was 

tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains 

TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA 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 solvent. The test article formed a clear brown solution in 

DMSO. In the initial toxicity-mutation assay, EXP1313100 was tested at 1.7, 5.4, 17, 52, 164, 

512, 1,600 and 5,000 μg/plate in triplicate in the presence and absence of S9. The highest 

concentration 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 

9
any 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 EXP1313100 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 

aberrations 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 512 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 adequate 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.

10
In 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 control substance formulations were administered at a dose 

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

The concentrations analyzed in the formulations and the stability of the formulations were 

acceptable.

In the dose range finding study 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 treated 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 polychromatic 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 not 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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