SRC TR

- 13

- 074

HPV3 TEST RULE DATA ADEQUACY REVIEW ( FINAL ): TOXICITY TO ALGAE AND COMBINED REPEATE D

- DOSE/ REPRODUCTIVE/DEVELOP MENTAL 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

Novem ber 14, 2013Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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i 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

................................ ................................ ........... 18Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

- 44

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1 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.regul ations.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 Inhibit i on 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 dat a 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 requirement s 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

.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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2 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 Cyanobacter ia, Growth Inhibition Test” (2006 ) referenced as Method C.3 of Commission Regulation ( EC) No

. 76 1 /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

.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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3 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 concur rently 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 belo w 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 concentration s of 0 o r 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 10 3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100

- 150 r pm) and constant i llumination at 24 ± 1 °C until the algal cell density was approximately 10 4

- 10 5 cells/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 w ith the addition of 500 mg/L of sodium bicarbonate to provide a sufficient supply of CO 2 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: “ A n 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 10 5 cells 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 10 4 cells per mL and [were considered to have] had no significant dilution effect on the fi nal test concentration.” [ Vryenhoef 2013, p. 13]Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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4 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 begin ning 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. 1 4]

“ 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]

“ EC x 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 betwee n the test and control groups. All statistical analyses were performed usin g the SAS computer software package (SAS 1999

- 2001). ” [ Vryenhoef 2013, p. 16] Additional l y, 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, a n EPA harmonized test guideline for algal toxicity.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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5  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 spe cific 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 EC 50 (growth rate) > 100 mg/L 96

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

96

- hour EC 50 (yield) > 100 mg/L 96

- hour NOEC (yield) = 100 mg/L

96

- hour EC 50 ( 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 9 8.6 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).Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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6 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 2 8%

... 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 h ours 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 9 6

- 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 1 0.5 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 CO 2 from the gaseous phase to the aqueous phase. In this situation CO 2 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 gi ven in the Test Guidelines.” [Vryenhoef 2013, p. 19

- 20]

CONCLUSIONS

The 96

- hr EC 50 was > 100 mg/L for growth rate, yield

, and biomass based on nominal

conce ntrations. The 96

- hour N OEC 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 T G 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 10 3 cells/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 s ection

- 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.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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7 REFERENCES

Vryenhoef, H. 2013

. CAS# 111

- 44

- 4: Algal inhibition test. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41201 182. Sponsor: Champion Technologies, Inc., Houston, TX.

Text in quotes is taken directly from Vryenhoef (2013).Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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8 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 w ith Reproduction/Develop mental 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 Repea ted

- Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test

– Rat

GLP compliant?: Yes

Year study performed: 201 2

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 BPFinal HPV 3 Test Rule Data Adequacy Review CAS RN 111

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9 Exposure duration: Non

- recovery m ales , recovery males and females : 42 days; Non

- recovery f emales: 41

- 45 days

Doses/concentrations tested: 0, 5, 15

, or 5 0 mg/kg

- bw/day (doses were selected b ased 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 (D ay 5 of age). ” [ Fulcher and Watson 2013, p . 29]

“Data were analysed using the decision tree from the Provantis TM Tables and Statistics Module... The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were as sessed 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 Tes t 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 si gnificant 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), param etric 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

- paramet ric 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/kg

bw/day, “initial effects on body weight gain and food consumption were appare nt, with overall gain for males remaining lower than control.” [Fulcher and Watson 2013, p. 18]Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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10 “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 dose s 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 c oncentrations 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) we re 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 e arly 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 an imals, 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 post

coitum, and any female that failed to mate was euthanized at the end of the study.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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11 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 ma croscopically.

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/W ater 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. F ood efficiency (the ratio of body weight change/dietary intake) was calculated for non

- recovery males (except during mating), for non

recovery 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: N on

- 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 t he following parameters: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo

- erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closur e, 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.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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12 Hematology and Clinical Chemistry Measurements: “H a ematological and blood chemical investigations were performed on five males and five females selected from each non

- recovery test and control grou p 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 treatment

free 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 le ucocyte count (WBC) , differential leu k ocyte 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 anim als ) were subjected to a full detailed external and internal necropsy. Organ weights of 5 parental non

recovery 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 cerv ix, and vagina

. The following tissues and organs were also histologically examined for all remaining non

recovery 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,Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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13 mammary gland, ovaries, pituitary gland, prostate gland, seminal vesicles, testes, uterus, uterine cervix, and vagina

.

“ For all non

- recovery females, t he uterus was examined for signs of implantation and the number of u terine 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 observ ed 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 c alculate 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 o ffspring w as 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 salivat ion 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 thisFinal HPV 3 Test Rule Data Adequacy Review CAS RN 111

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14 study, this finding was considered not to represent a systemic toxic effect of t he 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 < 0.0 5 ) 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 stati stically 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 high

dose 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 s ignificance [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 no t 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 a nimals 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 male s.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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15 F emales 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 a ctivity value s 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 ac tivity 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 aminotransfera se 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 treatm ent 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 observ ed 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.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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16 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, o ne mated female in each of the control, 5, and 50 mg/kg

- bw/day groups failed to ac hieve 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 major ity 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 partur ition 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 u nrelated to treatment.

There were no treatment

- related effects on corpora lutea and implantation counts, pre

- and post

implantation 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

w ithin 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 toxi city 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

.Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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17 STUDY RELIABILITY

[2 ] Reliable with restrictions

. T he study authors did not provide a justification for the use o f the vehicle (Arachis oil BP), as required by 40 CFR 799.9365 and OECD 422 test guidelines.

REFERENCES

Fu l cher, S. and Watson, P. 2013. CAS# 111

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- 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).Final HPV 3 Test Rule Data Adequacy Review CAS RN 111

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18 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