SRC TR-13-074

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL):

TOXICITY TO ALGAE AND COMBINED REPEATED-DOSE/ 

REPRODUCTIVE/DEVELOPMENTAL TOXICITY

Ethane, 1,1’-oxybis[2-chloro-, CASRN 111-44-4

Prepared by:

Ashlee Aldridge, Justine Von Runnen, Jennifer Rayner, Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

November 14, 2013
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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 1

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 1

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 2

D-1. Toxicity to Aquatic Plants ................................................................................................. 2

D-2. Repeated-Dose/Reproductive/Developmental Toxicity .................................................... 8

E. DATA ADEQUACY SUMMARY TABLE........................................................................... 18
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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates toxicological data for ethane, 1,1’-oxybis[2-chloro- (CASRN 111-44-4)

that were submitted by Champion Technologies, Inc. to EPA in response to the final test rule, 

“Testing of Certain High Production Volume Chemicals; Third Group of Chemicals” (76 FR 

65385, October 21, 2011; referred to herein as HPV3 test rule; available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0086 as of 

November 12, 2013).

The HPV3 test rule requires testing of CASRN 111-44-4 for the following toxicological 

endpoints: C6 (toxicity to algae) and F1 (combined repeated-dose toxicity study with the 

reproduction/developmental toxicity screening test).

Champion Technologies, Inc. supplied the following study reports from toxicological testing: 

 CAS# 111-44-4: Algal Inhibition Test, submitted for the C6 testing requirement (study 

report not available online as of November 12, 2013)

 CAS# 111-44-4: Oral (Gavage) Combined Repeat Dose Toxicity Study with 

Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 with 

Recovery Groups), submitted for the F1 testing requirement (study report not available 

online as of November 12, 2013)

These newly submitted data are evaluated for adequacy herein. No other toxicity data are 

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

The submitted data are evaluated for adequacy in Section D. Each study was summarized 

according to a standard format and assigned a Klimisch reliability code based on its scientific 

merit and conduct, using professional judgment. To determine whether data were adequate to 

satisfy the C6 and F1 endpoints, study details were compared to current standard test guidelines 

to establish whether the study met current testing requirements. If the data available for the 

endpoint were determined to be inadequate, a basis for that conclusion is supplied. A table 

summarizing data availability/adequacy for each required endpoint is included on the last page of 

this document (Section E).

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Data submitted by Champion Technologies, Inc. are adequate to satisfy the C6 (toxicity to algae) 

and F1 (combined repeated-dose toxicity with reproduction/development toxicity screening) 

testing requirements for CASRN 111-44-4 promulgated in the final HPV3 test rule.

There are no outstanding HPV test rule requirements for toxicity testing of CASRN 111-44-4.
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D. DATA FOR SIDS ENDPOINTS

D-1. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata (Vryenhoef, 2013)

Title: CAS# 111-44-4: Algal inhibition test 

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Ethane, 1,1’-oxybis[2-chloro-, CASRN 111-44-4

Remarks: Purity: 99.7%; clear colorless liquid; Batch No. 45296HKV

METHOD

Method/guideline followed: OECD TG 201, “Freshwater Alga and Cyanobacteria, Growth 

Inhibition Test” (2006) referenced as Method C.3 of Commission Regulation (EC) No.

761/2009; US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400, “Algal Toxicity, 

Tiers I and II” (1996)

Test type: Toxicity Test to Freshwater Algae – static conditions

GLP compliant?: Yes 

Year study was performed: 2013

Test species: Green algae, Pseudokirchneriella subcapitata (strain CCAP 278/4); obtained from 

the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Scottish 

Marine Institute, Oban, Argyll, Scotland.

Analytical monitoring: Yes 

Exposure duration: 96 hours

Concentrations tested: 

Range-finding test: Nominal concentrations of test material: 0, 0.1, 1, 10, and 100 mg/L. Mean 

measured concentration of test material for nominal concentration of 100 mg/L: 109 mg/L.

Definitive test: Nominal concentrations of test material: 0 and 100 mg/L. Mean measured 

concentration of test material for nominal concentration of 100 mg/L: 99.3 mg/L.
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Test method/conditions remarks: 

Preliminary Range-finding Test: 

Test substance concentrations in the definitive test were based on a preliminary test in which 

algae were exposed to the test item at nominal concentrations of 0.1, 1, 10, or 100 mg/L under 

static conditions for 96 hours. A control group was tested concurrently under identical 

conditions but not exposed to the test item. Two replicate flasks were used for each control and 

treatment group.

Test conditions (including culture medium, experimental chambers, and lighting) were the same 

as those described below for the definitive test.

Definitive Test: 

Based on the results of the range-finding test, a limit test was conducted for the definitive test. 

Exponentially growing algal cultures were exposed to nominal concentrations of 0 or 100 mg/L 

under static conditions for 96 hours. Testing was conducted using 6 replicates for each group. 

Concentrations were measured at 0 and 96 hours. 

Preparation of Algae Culture: “Prior to the start of the test sufficient master culture was added to 

approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell 

density of approximately 103cells/mL. The flasks were plugged with polyurethane foam 

stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant 

illumination at 24 ± 1 °C until the algal cell density was approximately 104-105cells/mL.” 

[Vryenhoef 2013, p. 11]

Culture Medium: “The culture medium used for the range-finding and definitive test was the 

same as that used to maintain the stock culture with the addition of 500 mg/L of sodium 

bicarbonate to provide a sufficient supply of CO2 and to counteract the increase in pH due to 

algal growth in an enclosed system...” [Vryenhoef 2013, p. 12]

Experimental Chambers: For both the range-finding and definitive test, “The test was conducted 

in 250 mL glass conical flasks each completely filled with test preparation and sealed with 

ground glass stoppers.” [Vryenhoef 2013, p. 12]

Preparation of Test Solutions: “An amount of test item (200 mg) was dissolved in culture 

medium and the volume adjusted to 2 liters to give a 100 mg/L stock solution. This stock 

solution was inoculated with algal suspension (32.4 mL)...” [Vryenhoef 2013, p. 13]

“The stock solutions and each of the prepared concentrations were inverted several times to 

ensure adequate mixing and homogeneity.” [Vryenhoef 2013, p. 12]

Exposure Conditions: “Pre-culture conditions [for the definitive test] gave an algal suspension in 

log phase growth characterized by a cell density of 6.18 x 105cells per mL. Inoculation of 2 

litres of test medium with 32.4 mL of this algal suspension gave an initial nominal cell density of 

1 x 104cells per mL and [were considered to have] had no significant dilution effect on the final 

test concentration.” [Vryenhoef 2013, p. 13]
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For both the range-finding and definitive tests, “The flasks were incubated... at 24 ± 1 °C under 

continuous illumination (intensity approximately 7000 lux) provided by warm white lighting 

(380- 730 nm) and constantly shaken at approximately 150 rpm for 96 hours.” [Vryenhoef 2013, 

p. 13]

Observations: Cell densities were determined from samples taken at 0 and 96 hours for the 

range-finding test and at 0, 24, 48, 72, and 96 hours for the definitive test using a Coulter® 

Multisizer Particle Counter. The pH was measured at 0 and 96 hours. The temperature within 

the incubator was recorded hourly.

Analytical Monitoring: Samples from the control and 100 mg/L (nominal) test chambers were 

taken at the beginning and end of the test (0 and 96 hours) and were stored at approximately 

-20°C prior to analysis. Concentrations of test item were analytically determined by gas 

chromatography (GC) using an external standard. “The test item gave a chromatographic profile 

consisting of a single peak... The test item described in the biological part of this study was also 

used as the analytical standard.” [Vryenhoef 2013, p. 31] The limit of quantitation was 0.099 

mg/L.

Evaluation of Data and Statistical Analysis: “The average specific growth rate over the test 

duration was calculated for each replicate control and test item vessel using the nominally 

inoculated cell concentration as the starting value rather than the measured starting value...” 

[Vryenhoef 2013, p. 14]

“In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for

the control cultures and the results examined in order to determine whether the growth rate

remained constant.” [Vryenhoef 2013, p. 14]

“ECx values were determined by inspection of the growth rate, yield and biomass integral data 

after 96 hours.” [Vryenhoef 2013, p. 16]

“A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 

1981) was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours 

for the control and the 100 mg/L test concentration to determine any statistically significant 

differences between the test and control groups. All statistical analyses were performed using 

the SAS computer software package (SAS 1999-2001).” [Vryenhoef 2013, p. 16] Additionally, 

Dunnett's multiple comparison procedure was used for comparing several treatments with a 

control.

Validation Criteria: “The results of the test are considered valid if the following performance 

criteria were met:

 The cell concentrations of the control cultures must increase by a factor of at least 

16 after 72 hours (OECD/EC Guidelines) and by a factor of at least 100 after 96 hours 

(EPA/OPPTS Guidelines).1

 

1 OPPTS 850.5400 (guideline cited by test report) does not specify a minimum increase in cell concentration of 

control cultures. The requirement for an increase by a factor of at least 100x after 96 hours appears to have been 

derived from OCSPP 850.4500, an EPA harmonized test guideline for algal toxicity.
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 The mean of the coefficients of variation of the section by section specific growth 

rates in the control cultures during the course of the test (0-24, 24-48 and 48-72 

hours) must not exceed 35% (OECD Guidelines).

 The coefficient of variation of the average specific growth rate in replicate control 

cultures after 72 hours must not exceed 7% (OECD Guidelines).

 The coefficient of variation of the cell density values in replicate control cultures 

must not exceed 20% after 72 and 96 hours (EPA/OPPTS Guidelines).1” [Vryenhoef 

2013, p. 16]

RESULTS

Endpoint value(s): Based on nominal concentrations:

96-hour EC50 (growth rate) > 100 mg/L

96-hour NOEC (growth rate) = 100 mg/L 

96-hour EC50 (yield) > 100 mg/L

96-hour NOEC (yield) = 100 mg/L

96-hour EC50 (biomass) > 100 mg/L

96-hour NOEC (biomass) = 100 mg/L 

Results remarks:

Range-Finding Test:

“Chemical analysis of the 100 mg/L test preparation at 0 and 96 hours... showed a measured test 

concentration of 109% of nominal was obtained indicating that the test item was stable over the 

test duration.” [Vryenhoef 2013, p. 17]

Growth rate, biomass, and yield of algal cultures were not inhibited at test concentrations of 

0.10, 1.0, 10 and 100 mg/L. Therefore, the definitive test was conducted as a limit test at 100 

mg/L.

Definitive Test: 

Measured concentrations of test substance at the nominal test concentration of 100 mg/L ranged 

from 98.6 to 100% of nominal; therefore, the results are based on nominal test concentrations 

only.

Growth rate, yield, and biomass of P. subcapitata were not inhibited by the presence of the test 

item at a nominal test concentration of 100 mg/L over the 96-hour exposure period. There were 

no statistically significant decreases (P ≥ 0.05) in growth rate, yield, or biomass as compared to 

the control at 72 and 96 hours.

 

1 OPPTS 850.5400 does not specify a requirement for coefficient of variation of cell density values. OCSPP 

850.4500 specifies that the coefficient of variation in control cultures should be less than 15% for yield and growth 

rate at test termination (96 hours). 
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The test was considered valid based on satisfaction of the validation criteria. “[T]he cell 

concentration of the control cultures increased by a factor of 83 after 72 hours and by a factor of 

130 after 96 hours... The mean coefficient of variation for section by section specific growth 

rate for the control cultures over the [first 3 days of the] test period (0-72 hours) was 28%... The 

coefficient of variation for average specific growth rate for the control cultures over the [first 3 

days of the] test period (0-72 h) was 2%... The coefficient of variation of the cell density values 

in replicate control cultures was 9% after 72 hours and 1% after 96 hours.” [Vryenhoef 2013, p. 

17-18]

No microscopic abnormalities were observed in any of the control or test cultures. “At the start 

of the test all control and test cultures were observed to be clear colorless solutions. After the 

96-Hour test period all control and test cultures were observed to be bright green dispersions.” 

[Vryenhoef 2013, p. 19]

Over the course of the study, the temperature was maintained at 24 ± 1°C. The pH ranged from 

7.5 to 7.7 at test initiation and from 10.5 to 10.6 at the end of the test (96 hours).

“The pH value of the control cultures... was observed to increase from pH 7.5 at 0 hours to pH 

10.5 at 96 hours. This increase was considered to be due to the amount of carbon dioxide 

required by the large number of algal cells in the log phase of growth... exceeding the transfer 

rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for 

photosynthesis and growth would be derived from bicarbonate in solution which results in an 

increase in the pH of the culture. The increase in pH after 96 hours was in excess of that 

recommended in the Test Guidelines (1.5 pH units after 96 hours). This was considered to have 

had no adverse effect on the results of the study given that the increase in cell concentration in 

the control cultures exceeded the validation criterion given in the Test Guidelines.” [Vryenhoef 

2013, p. 19-20]

CONCLUSIONS

The 96-hr EC50 was > 100 mg/L for growth rate, yield, and biomass based on nominal

concentrations. The 96-hour NOEC for growth rate, yield, and biomass was 100 mg/L, the 

highest concentration tested. 

STUDY RELIABILITY

[2] Reliable with restrictions. The pH value of the control cultures increased from 7.5 at 0 hours 

to 10.5 at 96 hours. This increase in pH exceeds that which is recommended in OECD TG 201 

(which states that the pH should not increase by more than 1.5 units during the test). According 

to the methods of Mayer et al. (2000), if sodium bicarbonate is added to the medium then the 

inoculum cell density should be reduced (e.g., 3 x 103cells/mL) and the pH should be adjusted to 

7.0 with HCl before test initiation. In this study, however, cell density was not reduced and the 

pH was only adjusted to 7.5. Furthermore, growth of control cultures appeared to slow after 48 

hours, as demonstrated by section-by-section growth rates of 0.074, 0.069, 0.043, and 0.019 

cells/mL/hour for exposure periods of 0-24, 24-48, 48-72, and 72-96 hours, respectively. 

Despite these deficiencies, growth in the control cultures satisfied the validation criteria.
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REFERENCES

Vryenhoef, H. 2013. CAS# 111-44-4: Algal inhibition test. Performing Laboratory: Harlan 

Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41201182. Sponsor: Champion 

Technologies, Inc., Houston, TX. 

Text in quotes is taken directly from Vryenhoef (2013).
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D-2. Repeated-Dose/Reproductive/Developmental Toxicity

Summary of repeated-dose endpoint: Adequate

Summary of reproductive toxicity endpoint: Adequate

Summary of developmental toxicity endpoint: Adequate

Study 1: Combined repeated-dose oral toxicity study with the reproduction/developmental 

toxicity screening test in rats (Fulcher and Watson, 2013)

Title: CAS# 111-44-4: Oral (Gavage) Combined Repeat Dose Toxicity Study with 

Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 with Recovery 

Groups)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Ethane, 1,1’-oxybis[2-chloro-, CASRN 111-44-4

Remarks: 99% purity; clear, colorless liquid; Batch No. 45296HKV

METHOD

Method/guideline followed: EPA 40 CFR 799.9365 TSCA guideline, “Combined Repeated 

Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”; OECD TG 

422 (1996), “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental 

Toxicity Screening Test”; Commission Regulation (EC) No. 440/2008

Test type: Combined Repeated-Dose Toxicity Study with Reproduction/Developmental 

Toxicity Screening Test – Rat

GLP compliant?: Yes

Year study performed: 2012

Test species: Wistar rats [Han:RccHan:WIST)]; male and female; source: Harlan Laboratories, 

U.K. Ltd., Blackthorn, Bicester, Oxon, UK; weight: 310-355 g (males) and 202-234 g (females); 

age: ~12 weeks old at initiation.

Number of animals/sex/dose: 12 (non-recovery group); 5 (recovery group)

Route of administration: Oral – Gavage

Vehicle: Arachis oil BP 
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Exposure duration: Non-recovery males, recovery males and females: 42 days; Non-recovery 

females: 41-45 days

Doses/concentrations tested: 0, 5, 15, or 50 mg/kg-bw/day (doses were selected based on 

available toxicity data including a fourteen day range-finding toxicity study in which body 

weight gain was lower in males at the 50 mg/kg bw/day dose level)1

Statistical methods used: “Where considered appropriate, quantitative data was [sic] subjected 

to statistical analysis to detect the significance of intergroup differences from control; statistical 

significance was achieved at a level of p < 0.05. Statistical analysis was performed on [mean 

values].” [Fulcher and Watson 2013, pp. 29-30]

“The standard unit of assessment was considered to be the litter, therefore values were first 

calculated for each litter and the group mean was calculated using the individual litter values. 

Group mean values included all litters reared to termination (Day 5 of age).” [Fulcher and 

Watson 2013, p. 29]

“Data were analysed using the decision tree from the ProvantisTM Tables and Statistics 

Module... The homogeneity of variance from mean values was analysed using Bartlett’s test. 

Intergroup variances were assessed using suitable ANOVA [Analysis of Variance], or if 

required, ANCOVA [Analysis of Covariance] with appropriate covariates. Any transformed 

data were analysed to find the lowest treatment level that showed a significant effect, using the 

Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose 

response was found, but the data shows [sic] non-homogeneity of means, the data were analysed 

by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant 

difference from the control group. Where the data were unsuitable for these analyses, pair-wise 

tests was [sic] performed using the Student t-test (parametric) or the Mann-Whitney U test 

(nonparametric).” [Fulcher and Watson 2013, p. 30]

“Data not analysed by the Provantis data capture system were assessed separately using the SPSS 

statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. 

Where Levene’s test was shown to be non-significant (p ≥ 0.05), parametric analysis of the data 

was applied, incorporating analysis of variance (ANOVA). If this data was [sic] shown to be 

significant, this analysis was followed by pair-wise comparisons using Dunnett’s test. Where 

Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the 

Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Dose 

response relationship was also be investigated by linear regression. Where the data was [sic] 

unsuitable for these analyses, then pair-wise tests were performed using the Student t-test 

(parametric) or the Mann-Whitney U test (non-parametric).” [Fulcher and Watson 2013, p. 30]

 

1 The 14-day range-finding study was Harlan Project No. 41201179. The study report for the range-finding study 

was not available for review, but a summary was provided in the study report for the repeated-dose study with 

reproductive/developmental toxicity screening. According to this summary, effects on body weight and food 

consumption were observed at 75 mg/kg-bw/day, leading to the early termination of one animal. At 50 mg/kgbw/day, “initial effects on body weight gain and food consumption were apparent, with overall gain for males 

remaining lower than control.” [Fulcher and Watson 2013, p. 18]
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“Due to the preponderance of non-normally distributed data, reproductive parameters 

(implantation losses, offspring sex ratio and offspring surface righting) were generally analysed 

using non-parametric analyses.” [Fulcher and Watson 2013, p. 30]

“Data for females during gestation and lactation, and offspring data were assessed for dose 

response relationships by linear regression analysis, followed by one way analysis of variance 

(ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were 

shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where 

Levene’s test showed unequal variances the data were analysed using non-parametric methods: 

Kruskal-Wallis ANOVA and Mann-Whitney U test.” [Fulcher and Watson 2013, p. 31]

Test method/conditions remarks: 

Analyses of Test Item Formulations:

The stability and concentration of prepared test item formulations were determined by gas 

chromatography. During the study, samples of the test item formulations (nominal 

concentrations of 0, 1.25, 3.75, and 12.5 mg/mL (corresponding to doses of 0, 5, 15, and 50 

mg/kg-bw/day, respectively) were collected within two days of preparation and analyzed by gas 

chromatography to verify exposure concentrations. Measured concentrations were ≥ 100% of 

nominal in all test item samples. Homogeneity of formulations was assessed visually, and 

formulations were deemed homogeneous by visual inspection. Analyses of test item 

formulations (nominal concentrations: 0.25 and 25 mg/mL) showed that 98-102% of initial 

concentrations remained after storage for 20 days in the dark at 4°C. Therefore, formulations 

were prepared weekly for the first two weeks and twice monthly thereafter and stored at 4°C in 

the dark.

Dosing and Environmental Conditions:

For non-recovery test animals, groups of rats (12/sex/group) were orally administered the test 

item daily via gavage at dose levels of 0, 5, 15, or 50 mg/kg-bw/day at a dosing volume of 4 

mL/kg-bw. The concurrent control group was treated with the vehicle only. Treatment 

continued throughout the pre-mating (14 days), mating (maximum of 14 days), and post-mating 

periods. Exposure continued in males and females until scheduled euthanasia, for total 

exposures of 42 consecutive days (males) or 41-45 consecutive days (females). Females were 

dosed through gestation and early lactation, but were not dosed during parturition, if applicable.

During the mating period, animals were paired (1 male: 1 female) until there was evidence of 

mating (i.e., the presence of sperm in a vaginal smear and/or the presence of a vaginal plug). 

Following evidence of mating (designated as day 0 post-coitum), males were returned to the 

original cage. Mated females were housed individually (or with their litters after delivery) 

throughout gestation and lactation. 

For the non-recovery test animals, parental males were euthanized on day 43. Parental females 

were allowed to deliver and rear their pups until postpartum (lactation) day 5, when all parental 

females and their pups were euthanized. Non-pregnant females were euthanized on day 25 postcoitum, and any female that failed to mate was euthanized at the end of the study.
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For the recovery test animals, two groups of rats (5/sex/group) “were treated with the high dose 

(50 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained 

without treatment for a further fourteen days.” [Fulcher and Watson 2013, p. 11] The recovery 

animals were not mated.

For the recovery group animals, males and females were sacrificed after the 14-day recovery 

period and examined macroscopically. 

Clinical Observations:

All animals were observed for overt signs of toxicity, ill-health, and behavioral changes 

immediately before dosing, and three times after each daily dose during the week. “Animals 

were observed immediately before dosing, soon after dosing, and one hour after dosing at 

weekends (except for females during parturition where applicable). During the treatment-free 

period, recovery animals were observed daily.” [Fulcher and Watson 2013, p. 20] 

Body Weight and Food/Water Consumption:

Individual body weights of parental males and recovery animals were recorded on Day 1 (prior 

to dosing), and at weekly intervals until the time of sacrifice. Individual body weights of 

parental females were recorded on Day 1 (prior to dosing), weekly until mating was apparent, 

and on Days 0, 7, 14, and 20 post-coitum, and on Days 1 and 4 postpartum. 

Food consumption was recorded weekly on a per-cage basis (males and recovery animals 

throughout the study; females during the pre-mating phase) or for post-coital weekly intervals for 

each female during the post-mating phase. Food consumption was also recorded on postpartum 

days 1 and 4 for females with live litters. Food efficiency (the ratio of body weight 

change/dietary intake) was calculated for non-recovery males (except during mating), for nonrecovery females (pre-mating only), and for recovery animals throughout the study period. 

Water intakes were measured daily during the first two weeks of the study.

Neurological Assessments:

Non-recovery animals were observed for signs of functional/behavioral toxicity prior to the start 

of treatment and at weekly intervals thereafter. Behavioral assessments, conducted for all of the 

non-recovery test animals, consisted of evaluation of the following parameters: gait, tremors, 

twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, 

exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, 

urination, defecation, transfer arousal, and tail elevation.

Functional performance and sensory reactivity tests were also performed on 5 parental

rats/sex/dose group prior to termination. Functional performance tests evaluated motor activity 

and forelimb/hindlimb grip strength, while sensory reactivity tests measured reactivity to 

auditory, visual, and proprioceptive stimuli. Measured sensory reactivity parameters included

grasp response, vocalization, toe pinch, touch escape, pupil reflex, blink reflex, startle reflex, tail 

pinch, and finger approach.
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Hematology and Clinical Chemistry Measurements:

“Haematological and blood chemical investigations were performed on five males and five 

females selected from each non-recovery test and control group prior to termination (Day 42 for 

males and Day 4 post partum for females). In addition, haematological and blood chemical 

investigations were performed on all recovery group animals after the fourteen day treatmentfree period on Day 56.” [Fulcher and Watson 2013, p. 24] 

“Urinalytical investigations were performed on five non-recovery males from the control and

each test group during the final week of treatment and on all recovery animals during the

final week of the recovery period.” [Fulcher and Watson 2013, p. 24] 

Measured hematological parameters included: hemoglobin, erythrocyte count (RBC), 

hematocrit, erythrocyte indices (mean corpuscular hemoglobin, mean corpuscular volume, mean 

corpuscular hemoglobin concentration), total leucocyte count (WBC), differential leukocyte 

count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, prothrombin 

time, and activated partial thromboplastin time.

Measured serum chemistry parameters included: albumin, total protein, albumin/globulin ratio, 

total bilirubin, urea, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate 

aminotransferase, glucose, total cholesterol, calcium, chloride, potassium, sodium, bile acids, and 

inorganic phosphorus.

Measured urine parameters included: volume, specific gravity, pH, protein, glucose, ketones, 

bilirubin, urobilinogen, blood, and appearance.

Gross Necropsy and Histopathology:

All animals (including non-recovery adults, their offspring, and recovery animals) were 

subjected to a full detailed external and internal necropsy. Organ weights of 5 parental nonrecovery rats/sex/dose group and all of the animals within the recovery dose groups were 

recorded for the adrenals, brain, spleen, heart, kidneys, liver, thymus, and thyroid with 

parathyroids. Additionally, organ weights were recorded for all animals for the epididymides, 

prostate, seminal vesicles, ovaries, pituitary gland, testes, and uterus with cervix.

The following parental tissues and organs were histologically examined for 5 non-recovery 

rats/sex at 0 and 50 mg/kg-bw/day: gross lesions, adrenal glands, aorta, bone and bone marrow 

(femur with stifle joint and sternum), brain (cerebrum, cerebral cortex, and medulla/pons), 

caecum, colon, duodenum, epididymides, eyes, heart, ileum (with Peyer’s patches), jejunum, 

kidneys, liver, lungs and bronchi, lymph nodes (mandibular and mesenteric), mammary gland, 

muscle (skeletal), esophagus, ovaries, pancreas, parathyroid gland, prostate gland, pituitary 

gland, rectum, salivary gland (submaxillary), sciatic nerve, seminal vesicles with coagulating 

glands, skin (hindlimb), spinal cord (cervical, lumbar, and thoracic), spleen, stomach, testes,

thymus, thyroid glands, trachea, urinary bladder, uterus, uterine cervix, and vagina. 

The following tissues and organs were also histologically examined for all remaining nonrecovery rats/sex at 0 and 50 mg/kg-bw/day, all infertile males of any dose group, and all 

females that did not produce a litter: all gross lesions, epididymides, coagulating glands, 
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mammary gland, ovaries, pituitary gland, prostate gland, seminal vesicles, testes, uterus, uterine 

cervix, and vagina.

“For all non-recovery females, the uterus was examined for signs of implantation and the number 

of uterine implantations in each horn was recorded... Where applicable, the number of corpora 

lutea externally visable [sic] for the ovaries was recorded.” [Fulcher and Watson 2013, p. 25-26] 

Histological examinations of the testes included evaluation of stage in the spermatogenic cycle in 

the seminiferous tubules and effects on cell types present within the different stages. 

Reproduction/Developmental Toxicity:

Pregnant females were observed daily and the following parameters were recorded: date of 

pairing, date of mating, date and time of observed start of parturition, and date and time of 

observed completion of parturition. Parameters recorded on and after postpartum day 0 and used 

to calculate litter responses included number of live and dead offspring born, number of live 

offspring on postpartum days 1 and 4, sex of the offspring on postpartum days 1 and 4, and 

individual offspring weights on postpartum days 1 and 4 (individual weights were used to 

retrospectively calculate litter weights). Clinical condition of the offspring was recorded from 

birth to postpartum day 5. Offspring surface righting reflex was evaluated on postpartum day 1.

Calculated reproductive parameters included: pre-coital interval, mating index, pregnancy index, 

gestation length, parturition index, pre- and post-implantation loss, live birth index, viability 

index, and sex ratio. 

RESULTS

Endpoint value(s): 

Systemic Toxicity: 

LOAEL (males) = 50 mg/kg-bw/day, based on reduced body weight gains during selected time 

periods of the study

NOAEL (males) = 15 mg/kg-bw/day

NOAEL (females) = 50 mg/kg-bw/day, based on no effects at the highest dose tested

Reproductive/Developmental Toxicity:

NOAEL = 50 mg/kg-bw/day, based on no effects at the highest dose tested

Results remarks:

Clinical Observations:

No parental animal mortalities occurred. “At 50 mg/kg bw/day, transient post-dosing salivation 

was observed for both sexes. This observation was first apparent from Day 15 of treatment for 

males and Day 16 of treatment for females and, although the incidence of the finding showed 

large individual variation, all animals were affected at some stage by termination.” [Fulcher and 

Watson 2013, p. 33]

“Post-dosing salivation is frequently observed when dosing via the oral gavage route and is often 

associated with distaste of the dosing formulations/test item. At the incidence observed on this 
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study, this finding was considered not to represent a systemic toxic effect of the test item.” 

[Fulcher and Watson 2013, p. 33]

Body Weight and Food/Water Consumption:

In the first week of the study, body weight gain in males of the non-recovery and recovery 

groups at 50 mg/kg-bw/day was significantly lower (p < 0.05) than controls; reductions in body 

weight gain of 56 and 72%, respectively, were observed. A corresponding reduction in body 

weight gain was observed in high-dose females during the first week of the study; however, this 

36% reduction, relative to controls, was not statistically significant. Body weight gain was also 

reduced in high-dose recovery males during days 22-29 (reduction of 36% relative to controls; p 

< 0.05). Despite these differences in body weight gain, there were no statistically significant 

differences in body weight of males or females throughout the study, with the exception of highdose females on postnatal day 1.

“At 50 mg/kg bw/day, lactation body weights [on postnatal day 1] were lower [by 7%] than 

controls, with differences attaining statistical significance [p < 0.05]. In the absence of an effect 

on overall body weight gain [during lactation] the difference from control was considered to be 

of no toxicological significance.” [Fulcher and Watson 2013, p. 34]

“At 50 mg/kg bw/day, a slight [but not significant] reduction in food efficiency was apparent for 

animals of either sex during the first week of treatment and for recovery males only during Week 

4, which was consistent with the lower body weight gain observed during these periods. 

Subsequent food efficiency was similar to control and the intergroup difference was considered 

to be of no toxicological significance.” [Fulcher and Watson 2013, p. 35]

Water consumption was not affected by treatment.

Neurological Analyses: 

“Weekly open field arena observations did not reveal any treatment related effects in treated 

animals when compared to controls... No toxicologically significant effects of treatment were 

detected in grip strength or motor activity assessments for treated animals when compared to 

controls... There were no toxicologically significant changes in sensory reactivity. ” [Fulcher 

and Watson 2013, p. 33-34] 

Hematology and Clinical Chemistry Measurements:

There were no toxicologically-relevant, treatment-related effects on hematology or clinical 

chemistry endpoints.

Non-recovery rats:

Mean neutrophil counts were significantly reduced (p < 0.01), compared with control, in all 

treated groups of males, but the decreases were not dose-related and the mean neutrophil count in 

control males was greater than the highest male historical control value. Mean activated partial 

thromboplastin time was significantly reduced (p < 0.05) in males at 50 mg/kg-bw/day, but this 

mean was within the historical control range for males. 
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Females at 15 and 50 mg/kg-bw/day showed a statistically significant increase (p < 0.05) in 

mean corpuscular volume (MCV) and a statistically significant reduction (p < 0.05) in mean 

corpuscular hemoglobin concentration (MCHC), relative to the control group; however, the 

mean MCV and MCHC values in these dosed groups were within historical control ranges for 

females. Females at 50 mg/kg-bw/day also showed a statistically significant reduction (p < 0.05)

in erythrocyte count (RBC) and a statistically significant increase (p < 0.05) in neutrophil count

(Neut), relative to the control group; however, the mean RBC and Neut values in these dosed 

females were within historical ranges for females. 

Significantly lower (p < 0.05) aspartate aminotransferase activity values were observed in males 

at 5, 15, and 50 mg/kg-bw/day compared with control, but the decreased activity values were not 

dose-related, mean activity values in the dosed groups were within the historical control range 

for males, and the mean activity value in control males was greater than the highest male 

historical control value. 

Compared with control, significant decreases in males were seen in blood urea (p < 0.01) and 

phosphorus levels (p < 0.05) at 50 mg/kg-bw/day and in blood potassium (p < 0.05) at 15 and 50 

mg/kg-bw/day. However, the mean levels for urea, phosphorus, and potassium levels in these 

dosed male groups were within historical control ranges for males. 

“For females at 50 and 15 mg/kg bw/day, higher alanine aminotransferase levels attained 

statistical significance compared to controls [p < 0.05]. Additionally for females at 50 mg/kg 

bw/day lower urea values attained statistical significance compared to controls [p < 0.05].” 

[Fulcher and Watson 2013, p. 38] However, the mean values for alanine aminotransferase 

activity and urea in these dosed female groups were within historical control ranges for females.

Recovery rats:

Following fourteen days without treatment, blood urea was significantly higher (p < 0.05) in 

recovery 50 mg/kg-bw/day males than in recovery controls; however, the mean urea 

concentration in the 50 mg/kg-bw/day recovery males was within the historical control range for 

males. 

“Assessment of urine parameters did not reveal any obvious effects of treatment for males at the 

end of the treatment period, or both sexes at the end of the recovery period.” [Fulcher and 

Watson 2013, p. 39]

Gross Necropsy and Histopathology:

Significant increases in absolute and relative thyroid/parathyroid weights were observed in males 

and females at 50 mg/kg-bw/day (p < 0.05), but the mean values at this dose were within the 

historical control range. No other significant differences in organ weights were observed.

Necropsy showed no treatment-related gross or microscopic lesions in males or females. 
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Reproductive/Developmental Toxicity:

No treatment-related effects were noted for fertility or mating performance. In each group, ten 

females gave birth to a live litter and successfully reared young to postnatal Day 5.

One control female and one female in the 15 mg/kg-bw/day group did not show any positive 

evidence of mating and did not produce a pregnancy. In addition, one mated female in each of 

the control, 5, and 50 mg/kg-bw/day groups failed to achieve pregnancy. These occurrences 

were considered incidental and unrelated to treatment. All other females mated successfully and 

gave birth to at least one pup.

“Gestation length was unaffected by treatment at 5, 15 and 50 mg/kg bw/day, with the majority 

of littering females showing a gestation length between 22 and 231⁄2 days.” [Fulcher and Watson 

2013, p. 36]

One female in each treatment group (i.e., 5, 15, and 50 mg/kg-bw/day) showed total litter loss 

postpartum. These females had an extended parturition length (24, 241⁄2, or 251⁄2 days). Two of 

these females (at 15 and 50 mg/kg-bw/day) had a low number of implantation sites (2 per 

female) and all three females delivered 2 or fewer pups at parturition. These occurrences were 

considered incidental and unrelated to treatment.

There were no treatment-related effects on corpora lutea and implantation counts, pre-and postimplantation loss, live birth index, postnatal litter size, offspring survival, sex ratio, pup body 

weight, total litter weight, pup body weight gain, or surface righting ability. At 5 mg/kg-bw/day, 

the viability index at Day 4 was significantly (p < 0.05) less than the control. This was most 

likely due to 12/14 pups lost in a single litter between postnatal Days 1 and 4. Other litters 

within the group lost no more than 1 pup. Female offspring exhibited significantly (p < 0.05) 

increased body weight, relative to controls, on Day 1 at 15 mg/kg-bw/day and on Day 4 at 5 

mg/kg-bw/day; however, no significant changes were observed in total litter weight at any time 

point. Therefore, the statistically significant changes in viability index and female pup body 

weight were considered incidental and unrelated to treatment.

Necropsy found no treatment-related external or internal gross lesions in pups. 

CONCLUSIONS

The LOAEL for systemic toxicity in males was 50 mg/kg-bw/day, based on reduced body weight 

gains during selected time periods of the study. The NOAEL for systemic toxicity in males was 

15 mg/kg-bw/day. The NOAEL for systemic toxicity in females was 50 mg/kg-bw/day, based 

on no effects at the highest dose tested.

The NOAEL for reproductive and developmental toxicity was 50 mg/kg-bw/day, based on no 

effects at the highest dose tested.
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STUDY RELIABILITY

[2] Reliable with restrictions. The study authors did not provide a justification for the use of the 

vehicle (Arachis oil BP), as required by 40 CFR 799.9365 and OECD 422 test guidelines.

REFERENCES

Fulcher, S. and Watson, P. 2013. CAS# 111-44-4: Oral (Gavage) Combined Repeat Dose 

Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 

422 with Recovery Groups). Performing Laboratory: Harlan Laboratories Ltd., Shardlow, 

Derbyshire, UK. Project No. 41201180. Sponsor: Champion Technologies, Inc., Houston, TX. 

Text in quotes is taken directly from Fulcher and Watson (2013).
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E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Toxicity to Aquatic Plants (Algae) Yes Yes No

Health Effects

Repeated-Dose Toxicity Yes Yes No

Reproductive Toxicity Yes Yes No

Developmental Toxicity Yes Yes No