EPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

RISK ASSESSMENT: PMN 07-0601

REFLECTING DELIBERATIONS AND DE CISIONS FROM THE 03/04/09 RAD DISPO

I. BACKGROUND

Structure: [ ]

Identity: 2,3,3,3-Tetrafluoro-1-propene CAS No : 754-12-1

Categorical Name : [ ] Formula: C 3 H 2 F 4

Concerns Identified at FOCUS: Developmental Toxicity

Submitter: [ ]

Site(s): [ ]; OEM MACs during auto Mfg = 11; Servicing of MACs by repair shops

and DIYs = thousands

Production Volume: [ ]

Use: [ ]

P/Chem Properties: Colorless gas; MW = 114.04; MP = - 152.2 o C (Beilstein); BP = - 28.3 o C (Beilstein); VP = 88 psia at 21.1 o C (Meas) = 4551 torr, 206 psia at 54.4 o C (Meas) = 10653 torr WS = 198.2 mg/L at 24 o C (Meas); Log K

ow : 2.15 (est)

Exposure Routes of Concern: Worker and Consumer via Inhalation

II. HEALTH ASSESSMENT SUMMARY

Background This is the second RAD disposition meeti ng on P07-601. At the first disposition meeting, there were questions about the rat deve lopmental toxicity study and whether the effects seen were significant. It was decided to evalua te a satellite on a 2 generation study being run by

the submitter to address this issue. The submitte r conducted a satellite to a 2 generation study in

Wistar rats to assess skeletal effects to address the reversibility of the wavy ribs. Assessment of the weanlings should also shed light on whether the difference in placen tal weights and “small fetuses” has any significance. If the ribs are normal in the weanli ng satellite to th is study it will indicate that either the wavy ribs were reversible or not repeatable and alleviate the concern for this endpoint. Exposures were to 0, 5,000, 15,000, and 50,000 ppm nose only 6 hours/day and 5 days per week for 10 weeks prior to mating, one week during mating and through day 19 of gestation.EPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

CONCLUSIONS ON INTERIM SACRIFICE IN RAT 2-GENERATION STUDY

RAD scientist, Ernest Falke, reviewed the contractor review (atta ched) of the interim report (1 st generation only) of inhalation 2-generation reproductive toxicity study in rats and recommends NOAELS for the following observa tions that may have been significant.

1. Significantly decreased sex rati o at 50,000 ppm with the Fisher’s exact test but not with covariate analysis. The NOAEL is 15,000 ppm.

2. Decreased relative and absolute thymus wei ghts at the low dose and relative weights at the high dose but not at the intermediate dose. There was no dose response to this effect

and is not consider ed compound related.

3. Significantly delayed mean time to vaginal opening in F1 females at 15,000 and 50,000 ppm. The NOAEL for th is effect was 5,000 ppm.

4. Some minor rib variations we re noted but the frequency was low, not statistically significant and not dose relate d. Therefore the changes are not considered compound

related.

NOAEL FOR MOE CALCUALTIONS INVOLVING MULTIPLE EXPOSURES: At the first RAD disposition meeting, the rabbit developm ental toxicity study was discussed. No deaths in damns or compound related effects were noted at 2,500 and 4,000 ppm and deaths occurred at 7,500, 10,000, and 50,000 ppm. The NOAEL for lethality in rabbits of 4,000 ppm can be used as

the POD in the derivation of an MOE since it is lower than the NOAEL noted in the 2 generation rat study interim sacrifice of 5,000 and the lethality seen in the rabbits is a more significant effect. The 4,000 ppm exposure will be adjusted to a human equivalent concentration (HEC) using the modeling previously submitted by the submitter and reviewed by EPA. The HEC will

be used in the MOE calculations.

The 4,000 ppm exposure was for 23 days and wi ll be used in MOE calculations involving multiple exposure scenarios. This type of e xposure is generally inappropriate for calculation MOEs from single exposures, espe cially where single exposure data are available. For this reason, rat studies discussed below will be used for MOE calculations for single exposures.

NOAEL FOR MOE CALCUALTIONS INVO LVING SINGLE EXPOSURES: [ ] conducted a 4 hour acute toxicity test of the PMN chemical, nose only, in Sprague Dawley rats involving 5 males a nd 5 females at each dose. Exposures were to 201,600 ppm and 405,800 ppm. Oxygen was maintained at 19%. Anim als were kept for 14 days after exposure and gross necropsy performed. No rats died but at necropsy “grey discol oured lungs” were seen in 1 female and 1 male of the low dose group an d 1 female and 3 males of the high dose group.

Authors could not discount th e lung effects as co mpound related since there was no control group. In the absence of any evidence to the contrary, 201,600 ppm will be considered a

LOAEL.EPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

[ ] conducted a two week inhalation toxicity study of the PMN chemical in Sprague Dawley Rats exposed for 6 hours a day, 5 days a w eek of two weeks. Analytically measured concentrations were 4,990, 19,599, and 51,690 ppm. Pathology and histopathology did not reveal any compound related effect s. The only effect observed wa s a slight increase in calcium levels at the two highest doses which was not compound related because there was no doseresponse and the values were well within the historical control ra nge. If no effects were seen with multiple exposures to 51,690 ppm, then a sing le exposure for 6 hours would also be without effect. Therefore, 51,690 ppm will be used as th e NOAEL for acute exposure calculations of the

MOE.

HUMAN EQUIVALENT CONCENTRATION (HEC) DETERMINATION The Human Equivalent Concentration is the exposure to huma ns which would have to be exposed to result in

the same blood levels seen in the rabbit at the rabbit NOAEL exposure.

HEC calculation for the multiple exposure rabbit study . The following will discuss the background for deriving a Human Equivalent Con centration (HEC) for th e NOAEL for lethality in a rabbit developmental toxicity study. The NO AEL for lethality is exposure to 4,000 ppm of the PMN for 6 hours per day for gravid days ( GD) 6-28. This should be used for the MOE calculations. The next highest dose which show ed lethality was 5,500 ppm with the first death occurring on GD13 after 8 exposures. Blood leve ls of the PMN are a better measure of dose than external exposure concentrations. The subm itter performed modeling of the exposure levels of the PMN vs blood levels in rabbits, rats, and humans. Based upon this modeling, it takes approximately two times (1.9 used – see below) the exposure concentration in humans to attain

the same blood levels in the rabbit. Thus the HEC for an exposure to 4,000 ppm in the rabbit is 7,600 ppm in the human.

This is based upon the measured blood-air part ition coefficients of the PMN in rabbits, rats and humans [ ]. Rat female, rabbit male, and female human blood to air partition coefficients were 0.076, 0.072, and 0.038 respectively. Rat and rabbit were essentially identical

and about twice the human values. These values we re integrated into a m odel to relate exposure to blood dose levels [ ]. Steady state is reached within minut es in animals and humans. Exposure values vs blood concentrati ons predicted from the model are:

Species Exposure ppm Blood conc (mg/L)

Female human 80,000 14.2*

Female human 400 0.0709

Pregnant rabbit 4,000 1.342

Rat 50,000 17.71**EPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

*If blood level were directly proportional to ex posure concentration, one would predict that 80,000 ppm exposure would give a blood level of 15.8 mg/L if one uses the 400 ppm human exposure results as a starting point. Good agreement with this hypothesis.

** If blood level were directly proportional to ex posure concentration, rat kinetics were the same as rabbit (reasonable since their blood air partition coefficients are similar), one would predict that 50,000 ppm exposure would gi ve a blood level of 16.78 mg/L if one uses the 4,000 ppm rabbit exposure results as a starting point. Good agreement with this hypothesis.

In determining the HEC for the 4,000 ppm NOAEL in the rabbit the ideal would have been for the submitter to run that specific exposur e model. However, since blood levels seem to be directly proportional to expos ure levels in the 400 ppm to 80,000 ppm range it is reasonable to use this relationship to predic t the HEC at 4,000 ppm exposure. Since there is good agreement with the blood level being directly proportional to the exposure level, one would predict from the 400 ppm human female modeling results that 4,0 00 ppm would result in blood levels of 0.709 in the human female. The ratio of 1.34 (female rabbit blood level at 4,000 ppm) to predicted human blood levels at 4,000 ppm of 0.709 is 1.9. Using this crude approximation and the fact that blood levels are proportional to exposures, the HEC to give a blood level in the human of 1.34 mg/L (the NOAEL for lethality in the rabbit study) is 1.9 * 4,000 ppm = 7,600 ppm. This

should be used as the POD for the MOE calculations.

HEC calculation for the sing le exposures in the rat . Since the rat kinetics are very similar to the rabbit, the same factor of 1.9 derived above will be used to derive the HEC MOE for single exposure calculations from the rat NOAEL of 51,690 ppm. This is 1.9 * 51,690 ppm = 98,211

ppm.

AREA UNDER THE CURVE (A UC) CONSIDERATIONS

The submitter uses AUC considerations to pred ict short duration high level exposures. The 4,000 ppm blood levels in the rabbit developmenta l study were used to support this conclusion (minor point – submitter said study was for 22 days but exposure was from GD 6-GD28 which should be 23 days). They used blood levels of 1.342 mg/L for 6 hours per day for 22 days to give a total AUC of 177.3 mg/L -hr (1.342*6*22). They stated that in a 5 minute exposure to 80,000 ppm in a human would result in blood leve ls of 14.2 mg/L with an AUC value of 1.2 mg/L-hr or about 148 fold lower than the rabbit NOAEL AUC. If true th is type of calculation would indicate an adequate MOE. While the conclusion might have some merit (see below)

there are still uncertainties w ith this type of extrapolati on. The results of the rabbit developmental toxicity studies raise some intere sting questions. The e xposures and AUC for the exposure which caused death are summarized below:

Concentration ppm # exposures blood level AUC to first death mg/L mg/L-hr

*estimatedEPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

4,000 no deaths 1.342 177

in 22

exposures

5,500 8 1.85* 89 7,500 7 2.52* 106 10,000 7 3.36* 141 50,000 5 17.71 531

If the AUC argument really held, then one would predict that if 177 mg/L-hr is without effect that AUC values of 89, 106, and 141 should not have resulted in death. Yet death was observed for the 5,500, 7,500, and 10,000 ppm exposures with these respective AUC values. The situation is a complicated interweaving of concentration and time. As exposure concentration increases the AUC required to cause death increases. It seems that higher doses require a higher AUC to

cause an effect and that the blood levels caused by 4,000 ppm exposure in the rabbit can be sustained for a long time with no effect. Above 4,000 ppm some factor of time enters into the complex parameters surrounding the toxicity of the PMN.

From this analysis it appears th at 4,000 ppm is a NOAEL for lethality in the rabbit and that AUC calculations can be done for high le vel, short exposure durations but there is a moderate degree of uncertainty regarding the conclu sions one can draw about safety.

III. RISK ASSESSMENT – HEALTH

Toxicity data: Rabbit developmental toxicity study w ith exposures from gravid day GD 6-28 for 6 hours per day for 13 total exposures. The NOAEL is 4,000 ppm and the Human Equivalent Concentration (HEC) which will be use fo r MOE calculations is 7,600 ppm. There are

uncertainties about the relative contributions of duration of exposure and exposure duration to the toxicity. For exposure scenarios between 2 to 8 hours the HEC of 7,600 ppm will be divided by the predicted exposures to derive the MOE. Th is is a reasonable scien tific compromise given

the uncertainties involved. Other uncertainties involve extrapol ating from a 13 day exposure in an animal to 250 days per year exposure in a human.

Acute exposure MOEs will be derived from the HEC determine from the rat studies discussed above. The HEC of 98,211 ppm will be divided by the predicted exposures to derive the MOE.

MOEs are expressed as 2 significant figures.

MOE value – 30 vs 100 . The normal MOE is 100. This is generally a 10 to account for interspecies differences and a 10 to account for variability wi thin the human population. These factors each comprise two s ub-factors – a pharmacokinetic component and a pharmacodynamic component which by convention are considered roughly equal. The pharmacokinetic componentEPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

is related to differences in absorption and metabolism between and within species and the pharmacodynamic component is related to differen ces in which species and individuals respond to the compound in a toxic response. The HE C is calculated using pharmacokinetic modeling which removes the uncertainties of the pharmac okinetic component when extrapolating from animals to humans. For this reason the interspe cies uncertainty should be reduced from 10 to approximately 3. The total MOE would then beco me 3 (for interspecies differences) * 10 (for variability within the human population) = 30.

In the case of this PMN, and MOE of 30 is recommended.

Consumer exposure scenario: Leakage of PMN into passenger car compartment from evaporator - the rat HEC of 98,211 for single exposure will be used in MOE calculations.

Multi-Chamber Concentration and Ex posure Model (MCCEM) USEPA modeling : compact car
of 2.5 m 3 volume, ACH 0.3, car at rest, AC on full recirculation, fan on, refrigerant charge ≈ 1 kg. Modeled as single chamber. Assumptions from USEPA 2005.

Scenario 1 : large leak, 5 minutes @ 100 g/min (loss of one-half charge) [ ] Results: single event dose, 7194 mg; av erage event concen tration, 86,328 mg/m 3 (18,516 ppm); Peak concentration, 1.75 E5 mg/m 3 (37,500 ppm) Number of U. S. exposures/yr: 164 to 3,114 from l eak due to collision; 1 to 8 from non-collision

leak [ ]

MOE: The duration of exposure was not stated but will be assumed to be 30 minutes exposure since this is what the industry used in thei r model. A TWA of 18,516 pp m will be assumed for a 30 minute exposure.

MOE = (98,211 pppm / 18,516 ppm) = 5.3

Scenario 2 : medium leak, 5 minutes @ 3g/min [ ] Results: single event dose, 216 mg; average event concentration, 2,592 mg/m 3 (556 ppm); Peak concentration, 5.2 E3 mg/m 3 (1,100 ppm); Number of U. S. exposures/yr: 7 to 1,157 fr om sudden leak; 55 to 11,690 from leak following recharge [ ]

MOE: The duration of exposure was not stated but will be assumed to be 30 minutes exposure since this is what the industry used in their model. A TWA of 556 ppm will be assumed for a 30 minute exposure.

MOE = (98,211 ppm / 556 ppm) = 180

Consumer exposure scenario: DIY ScenariosEPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

Scenario 1 : Upright can position – 3,264 ppm MOE: The duration of exposure was not stated but will be assumed to be 30 minutes exposure since this is what the industry used in thei r model. A TWA of 3,264 pp m will be assumed for a 30 minute exposure.

MOE = (98,211 ppm / 3,264 ppm) = 30

Scenario 2 : Upside down can position – 45,000 ppm MOE: The duration of exposure was not stated but will be assumed to be 30 minutes exposure since this is what the industry used in thei r model. A TWA of 45,000 pp m will be assumed for a 30 minute exposure.

MOE = (98,211 ppm / 45,000 ppm) = 2.2*

* Note that with a divisor 45,000, in order to ob tain an MOE of 30, you would have to expose an animal to 1,350,000 ppm. It is not possible to expose animals to concentrations this high because they would suffer from anoxia before s howing any chemical-related effects.EPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

Industrial exposure scenarios:

MOE calculated by dividing 7,600 ppm by the exposure ppm for multiple exposure scenarios and dividing 98,211 ppm by the exposure ppm for the single exposure sc enario (Sudden large leak that technician is not aware of).

Supply chain
component Scenario # sites # workers Days/yr Hrs/day Ppm MOE 7,600/

exposure Manufacturing Liquid sampling 3 2 250 d/y

2 h/d 3.3* 2,300

Manufacturing Loading liquid raw

material into

containers 3 24 250 d/y

8 h/d 20** 380

Use: [ ] for

OEM MACs OEM MAC charging during auto manufacturing 11 220 250 d/y

8 h/d 105***

52.5**** 72

140

Use: Servicing

of MACs Exposure due to equipment

leaks

PV: Submitter

PV for

aftermarket - 25 250 d/y

8 h/d 3.94 1,900

Use: Servicing

of MACs Exposure due to equipment

leaks

PV: what if

NCS saturates

market - 329,000 250 d/y

8 h/d 3.94 1,900

Use: Servicing

of MACs Sudden large

leak that

technician is

not aware of

PV: what if

NCS saturates

market - 980 1 d/y 141 5 min

122 30

min 700 Single

exposure

810 single

exposure

* 8 hour TWA ** 20 average and 73 maximum

*** reasonable worst case   likely exposureEPA SANITIZED

TI: Maggie Johnson 564-8924 R AD Disposition Meeting: 03/04/09 NM: Audrey Binder 564-9033

Risk Assessment Co-Author: Ernest V. Falke 564-7646

IV. TEST RECOMMENDATIONS

The single exposure MOEs were calculated from ra t data because there we re no single exposure data on the pregnant rabbit, the most susceptibl e group. There is some uncertainty about whether toxicity would be seen in a rabbit from a singl e exposure to 50,000 ppm. For that reason a rabbit acute toxicity study can be conducte d to address this question. Rabbits should be exposed for 1 hour (acute exposure scenarios are for 5 to 30 mi nutes) using the OECD 403 protocol. Pregnant rabbits should be exposed on GD 12 (this is within the time-frame that pregnant rabbits started dying). Exposure concentrations of 10,000, 50,000, and 100,000 ppm should be used.

ATTACHMENT

Review of Interim Report (1 st Generation Only) of Inhala tion 2-generation Reproductive Toxicity Study in Rats for P-07-0601

REFERENCES:

Review of Interim Report (1 st Generation Only) of Inhala tion 2-generation Reproductive Toxicity Study in Rats for P-07-0601.Last updated 10-30-07 REVIEW OF DEVELOPMENTAL TOXICITY STUDY FOR P-07-0601

I. INTRODUCTION

This report summarizes the potential adverse developmental effects of PMN P-07-0601 based on results of an inhalation prenatal developmental toxicity study in rats with 2,3,3,3- tetrafluoropropene (“HFO- 1234yf ,” CAS RN 754-12-1).

II. CONCLUSIONS

The developmental toxicity LOAEL is 5000 ppm (the lowest concentration tested) based on the statistically significant and concentration-related finding of wavy ribs at all test concentrations. Increased placental weights and increased incidence of small fetuses/litter werealso seen at concentrations as low as 5000 ppm, and these findings may also be treatmentrelated. The maternal toxicity NOAEL is 50,000 ppm, the highest concentration tested.

III. BASIS FOR CONCLUSIONS

Groups of female mated Wistar rats (25 rats/sex/group) were exposed via nose-only inhalation to HFO-1234yf (99.9% purity) at target test concentrations of 0 (clean air), 5000, 15,000, or 50,000 ppm for 6 hours/day from gestation days (GD) 6 through 19. Mean actual daily test concentrations were 0, 5000, 15,106, and 50,315 ppm. Clinical signs were recorded twice daily, body weights were measured on GD 0, 3, 6, 9, 12, 15, 19, and 21, and food consumption was measured over the peri ods GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-19, and 19-21. Dams were sacrificed on GD 21 and were examined for gross abnormalities. Maternal tissues showing macroscopic abnormalities were removed and fixed in formaldehyde for possible microscopic examination. The uteri (including fetuses), ovaries, and placentas of all females were examined for the following parameters: number of corpora lutea; number of implantation sites; number of early and late resorptions; number of live/dead fetuses; fetal sex; number of grossly malformed fetuses and fetuses with external abnormalities; ovarian weights; uterine weights; fetal weights; placental weights; gro ss evaluation of the placentas. The followingreproductive performance parameters were also evaluated: female fecundity index; preimplantation loss; post-implantation loss; gestation index; fetal sex ratio.

Half of all fetuses in each litter were examined for soft tissue abnormalities and half were

stained with Alizarin Red S and examined for skeletal abnormalities.

There were no deaths, no significant treatment-related clinical signs, and no gross postmortem findings in dams. Mean maternal body weights indicated statistically significant body weight loss during GD 6 through 9 in the lo w-, mid-, and high-concentration groups, accompanied by significantly decreased food consumption during this period; however, this effect on body weight change was compensated during GD 9 through 12, during which the mean maternal body weight changes were significantly increased. Significantly increased foodLast updated 10-30-07 consumption was seen during GD 15 through 19 in the low- and mid-concentration groups, and significantly decreased food consumption was s een in the high-concentration group during GD 6 through 19. No clear concentration-related e ffects on food consumption or body weight change

were observed and therefore these effects are not considered adverse treatment-related effects.

There were no significant effects on fec undity or gestation indices, numbers of implantations, pre- and post-implantation losses, resorptions, live fetuses, or mean sex ratio atany test concentration. The number of corpora lutea was significantly increased in the midconcentration group only; because no concentration-response was evident, this effect was not

considered an adverse treatment-related effect.

Mean dam carcass weight (carcass weight: terminal body weight minus gravid uterine weight) was significantly increased in the mi d-concentration group from GD 6 compared with the control; carcass weight was also increased in the high-concentration group, but this effect was not statistically significant at this exposure level. Because this effect did not achieve statistical significance in the high-concentration gr oup, this effect was not considered an adverse

treatment-related effect. There were no eff ects on uterine or ovarian weights.

The incidence of small fetuses per litter was significantly increased in the mid- and highconcentration groups compared with the control [incidences (%) were 0/24 (0%), 3/21 (14%), 5/24 (21%), and 7/25 (28%) at 0, 5000, 15,000, and 50,000 ppm respectively]. The study

authors indicate that this is not an adverse e ffect, since there was no significant effect on fetal weights. However, the finding of increased incidence of small fetuses/litter cannot be dismissed

as non-treatment-related.

Mean placental weights were significantly increased in the low-, mid-, and highconcentration groups compared with the control (weights in grams were 0.4547±0.01047, 0.5413±0.01772, 0.5434±0.01502, and 0.5378±0.01125 at 0, 5000, 15,000, and 50,000 ppm respectively). The study report indicates that since no effects were seen during external observations of the placenta and since no effects were seen on fetal weights, the effect on placental weight is not considered to be an adverse effect. However, because no microscopic examinations of placental tissue were performed to determine whether or not pathology was present, the finding of increased placental weight s cannot be dismissed as non-treatment-related.

A concentration-related, statistically significant increase in the litter incidence of two or more wavy ribs was seen in fetuses in the low-, mid-, and high-concentration groups compared with control. The litter incidences (%) were 6/24 (25%), 17/21 (81%), 18/24 (75%), and 24/25 (96%) at 0, 5000, 15,000, and 50,000 ppm respectively. The study report concluded that this is not an adverse effect, based on a 1994 literature review performed by A. Kast (1994) on published data from 1982 through 1993 which indicated that wavy ribs in rats develop between gestation days 17 and 20 and “are more or less a completely reversible finding.” In contrast, however, Kast’s review of 74 scientific papers indicated that “Most authors consider wavy ribs as a malformation,” a conclusion that the authors of the prenatal developmental toxicity study of P-07-0601 seem to have ignored. In addition, findings such as wavy ribs are considered1 SRC consulted EPA developmental t oxicologist Jennifer Seed (EPA R AD), who indicated on 10/15/2007 (via email) that wavy ribs are considered a developmental effect regardless of whether they are reversible. In addition, EPA reproductive/developmental toxicology e xperts Elaine Francis and Kathy Anitole indicated (via e-mails on 10/25/2007 and 10/26/2007, respectively) that wavy ribs are considered a sign of developmental toxicity.

Last updated 10-30-07 treatment-related by EPA regardless of their reversibility. 1 Kast (1994) indicated that numerous compounds are known to cause wavy ribs in fetuses when administered to pregnant rats but that wavy ribs are rarely observed in fetuses of untreated dams (0.0% occurrence in 13 of 22 papers reviewed by Kast, 1994). In fetuses of dams treated with the test compounds noted in Kast’sreview, the incidence of wavy ribs may be moderately or extremely increased in a dosedependent manner. Furthermore, the dose-related, significant increase in wavy ribs seen in the prenatal developmental toxicity study with P-07-0601 was seen at a litter incidence as high as 24 in the absence of clear maternal toxicity. These points support the position that the wavy ribs finding should not be discounted as a non-treatment-related, non-adverse effect.

In addition to the clear dose-response for wavy ribs, several variations in bone ossification in fetuses were observed (on a per litter basis) and are listed below. These variations included both increases and decreases in ossification with no clear pattern, and therefore do not appear to constitute a clear adverse effect. • significantly increased litter incidence of incompletely ossified supra occipital skull bones in the high-concentration group compared with control [incidences (%) were 0/24 (0%), 2/21 (10%), 4/24 (17%), and 8/25 (32%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly increased litter incidence of incomplete ossification of 3 or more sternebrae in the mid- and high-concentration groups compared with control [incidences (%) were 1/24 (4%), 6/21 (29%), 8/24 (33%), and 9/25 (36%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly increased litter incidence of incomplete ossification of one or two cervical bodies in the low-, mid-, and high-concentration groups compared with control [incidences (%) were 17/24 (71%), 21/21 (100%), 24/24 (100%), and 25/25 (100%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly decreased litter incidence of incomplete ossification of three or more cervical bodies in the low-, mid-, and high-concentration groups compared with control [incidences (%) were 23/24 (96%), 7/21 (33%), 9/24 (38%), and 6/25 (24%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly decreased litter incidence of one or two unossified cervical bodies in the low-, mid-, and high-concentration groups compared with control [incidences (%) were 23/24 (96%), 3/21 (14%), 3/24 (13%), and 4/25 (2%) at 0, 5000, 15,000, and 50,000 ppm respectively]Last updated 10-30-07 • significantly increased litter incidence of three or more unossified cervical bodies in the mid- and high-concentration groups compared with control [incidences (%) were 19/24 (79%), 20/21 (95%), 24/24 (100%), and 25/25 (100%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly increased litter incidence of one or two unossified caudal bodies in the low-, mid-, and high-concentration groups compared with control [incidences (%) were 4/24 (17%), 14/21 (67%), 15/24 (63%), and 15/25 (60%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly decreased litter incidence of one or two incompletely ossified metatarsals in the low-, mid-, and high-concentration groups compared with control [incidences (%) were 24/24 (100%), 16/21 (76%), 19/24 (79%), and 19/25 (76%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly increased litter incidence of one or two unossified metatarsals in the low-, mid-, and high-concentration groups compared with control [incidences (%) were 6/24 (25%), 15/21 (71%), 19/24 (79%), and 21/25 (84%) at 0, 5000, 15,000, and 50,000 ppm respectively]

• significantly decreased litter incidence of one to four incompletely ossified phalanges in the low-, mid-, and high-concentration groups compared with control [incidences (%) were 20/24 (83%), 7/21 (33%), 12/24 (50%), and 11/25 (44%) at 0, 5000, 15,000, and 50,000 ppm respectively]

The developmental toxicity LOAEL is 5000 ppm (the lowest concentration tested) based on the statistically significant and concentration-related finding of wavy ribs at all test concentrations. Increased placental weights and increased incidence of small fetuses/litter werealso seen at concentrations as low as 5000 ppm, and these findings may also be treatmentrelated. The maternal toxicity NOAEL is 50,000 ppm, the highest concentration tested.

IV. ASSESSMENT OF STUDY QUALITY

The study reportedly followed OECD TG 414. The study appears to have adequately followed this guideline with the exception that the inhalation route of exposure was used instead of oral exposure. The study report indicated that the inhalation route was chosen because this is the most likely route of human exposure under the specific conditions of this chemical’s use.

V. REFERENCES

P-07-0601. Prenatal developmental inhalati on toxicity study with HFO-1234yf in rats. April 17, 2007.

Kast, A. 1994. Wavy Ribs. A Reversible Pathol ogic Finding in Rat Fetuses. Exp ToxicolLast updated 10-30-07 Pathol 46(3): 203-210.

OECD TG 414. Pre-natal Developmental Toxicity Study. OECD Guideline for Testing of Chemicals. Adopted January 22 2001.Last updated 1-22-09 REVIEW OF INTERIM REPORT (1 st GENERATION ONLY) OF INHALATION 2-

GENERATION REPRODUCTIVE TOXICI TY STUDY IN RATS FOR P-07-0601

I. INTRODUCTION

This report summarizes results presented in an interim report on the first generation of an inhalation 2-generation reproductive toxicity study in Wistar rats with 2,3,3,3-tetrafluoropropene ( A HFO-1234yf, @ CAS RN 754-12-1), submitted to EPA in September 2008.

II. CONCLUSIONS

As this is an interim report, no definitive conclusions can be made at this time. Based on the results for F0 parents 1 and F1 pups presented in this interim report, parental toxicity was observed at all test concentrations (reduced body weights, reduced food consumption, decreased organ weights) and toxicologically relevant, sta tistically significant changes seen in reproductive and/or developmental parameters were as follows:

 Significantly increased mean pre-coital time for F0 parents at all test concentrations (2.27, 3.44, 3.39, and 3.5 days at 0, 5000, 15,000, and 50,000 ppm respectively); however, all females (except 2 at 0 ppm and 1 at 5000 ppm) were mated within a week, and therefore the study report indicates that this finding was not relevant

 Significantly increased mean gestation duration in F0 dams at 5000 and 50,000 ppm (21.56, 21.92, 21.86, and 21.92 days, respectively, at 0, 5000, 15,000, and 50,000 ppm); however, the study report indicated that since gestation never exceeded 22 days, and since no abnormalities were noted during parturition, this finding was not relevant

 Significantly decreased sex ratio (percentage of male pups) for F1 pups at 50,000 ppm (53, 52, 44, and 41% respectively at 0, 5000, 15,000, and 50,000 ppm); the study report indicates that this was statistically significant at 50,000 ppm with the Fisher’s exact test but not with covariate analysis

 Significantly decreased absolute and relative thymus weights of F1 pups (both sexes) at 5000 ppm and significantly decreased relative thymus weights of female F1 pups at 50,000 ppm

 Significantly delayed mean time to vaginal opening in F1 females at 15,000 and 50,000 ppm (39.36, 40.36, 42.48, and 43.46 days at 0, 5000, 15,000, and 50,000 ppm respectively)

In F0 animals, there were no effects on sperm count, motility, or morphology; estrous cycling; mating/fertility/gestation indices; or post-implantation loss. In F1 pups, there were no effects on mean live litter sizes, mean and total numbers of pups born, pup weights, or pup

1 Microscopic evaluation of tissues has not yet been performed.Last updated 1-22-09 survival. There were no test substance-related macroscopic findings at necropsy in F1 pups and no significant skeletal observations.

III. BASIS FOR CONCLUSIONS

An interim report on an OECD TG 416- and OPPTS 870.3800-compliant 2-generation inhalation reproductive toxicity study in Wistar rats was provided by the submitter. The interim report covered the first-generation results only (the F0 generation and the F1 generation until weaning, excepting microscopic analyses of F0 tissues). Target vapor concentration levels were 0, 5000, 15,000, or 50,000 ppm. 2 F0 rats (28/sex/group) were exposed nose-only (for 6 hours/day, 5 days/week) for 10 weeks pre-mating, and for up to 1 week during the mating phase. During 3 week pre-mating, vaginal smears were made daily for all females (but only the smears from the 0 and 50,000 ppm groups were evaluated). F0 males were sacrificed after mating. F0 females were exposed nose-only (6 hours/day) th rough day 19 of gestation, and then again via whole-body inhalation (6 hours/day) from lactati on day (LD) 5 through 21. All F0 females were allowed to deliver naturally and rear their young to weaning (LD 21). On post-natal day 4, litters of more than 8 pups were adjusted by randomly eliminating extra pups to yield 4 males and 4 females per litter. On or shortly after LD21, F0 dams were sacrificed and some F0 offspring (F1) were selected for the next phase of the study (28 pups/sex/group). In these selected pups, preputial separation was recorded in males from post-natal day 39 onwards, and vaginal opening was recorded in females from post-natal day 31 onw ards. Other F0 offspring (F1) were selected for necropsy (1 pup/sex/litter). From these pups, the brain, thymus, spleen, and any organs/tissues showing macroscopic abnormalities were preserved. Brain, thymus, and spleen were weighed. These pups were also examined for skeletal abnormalities of the ribs and

sternum.

Parameters assessed for adult F0 animals included clinical signs, body weights, and food consumption. At necropsy of F0 males and fe males, the following organs were preserved: adrenals, brain, epididymides, kidneys, liver, ovaries including oviduct, pituitary gland, prostate, seminal vesicles and coagulating glands, spl een, testis, thyroid, uterus, vagina, and any organs/tissues showing macroscopic abnormalities. All but the vagina were weighed. In addition, analyses of epididymal sperm motility, count, and morphology were performed on all males from all groups. Reproductive and developmental parameters (including fertility, mating,days between initiation of mating and coitus, gestation, estrous cycling, pup mortality, postimplantation loss, mean live litter sizes, number of pups born, percentage of males per litter at birth, and postnatal survival) were evaluated for the F0 generation.

F0 parents – clinical signs, body weights, and food consumption: There were no test substance-related deaths or clinical signs. During pre-mating, mean body weights in males were statistically significantly decreased from weeks 7 through 11 at all three test concentrations, and additionally at 50,000 ppm during weeks 5 and 6 and at 15,000

2 The study report indicated that actual test concentrations were measured by total carbon analysis, and that details will be included in the final report.Last updated 1-22-09 ppm during weeks 2-6. In females during pre-mating, mean body weights were statistically significantly decreased at 50,000 ppm from weeks 1 through 10 and additionally at 15,000 ppm during weeks 3 and 9 only. Mean body weights of females were statistically significantly decreased at 50,000 ppm on GD 14 and LDs 1, 4, and 7, as well as at 15,000 ppm on LD1. During pre-mating, mean food consumption (in g/animal/day) in F0 males was statistically significantly decreased at 50,000 ppm during w eeks 6-7 and 9-10, at 15,000 ppm during weeks 2-3, 8-9, and 9-10, and at 5000 ppm during weeks 8-9 and 9-10. During pre-mating, mean food consumption (in g/animal/day) in F0 females was statistically significantly decreased at 50,000 ppm during weeks 2-3 and 5-6, and at 15,000 ppm during weeks 2-3. There were no effects on food consumption in g/kg/day. There were no significant effects on food consumption during gestation. During lactation, mean food consum ption (in g/animal/day and g/kg/day) was statistically significantly increased at 5000 ppm during days 1-4.

F0 parents – reproductive parameters:There were no effects on estrous cycling, mating/fertility/gestation indices, or postimplantation loss. The mean pre-coital time was statistically significantly increased at all test concentrations (2.27, 3.44, 3.39, and 3.5 days at 0, 5000, 15,000, and 50,000 ppm respectively); however, all females (except 2 at 0 ppm and 1 at 5000 ppm) were mated within a week, and therefore the study report indicates that this fi nding was not relevant. Also, mean gestation duration was statistically significantly increased at 5000 and 50,000 ppm (21.56, 21.92, 21.86, and 21.92 days, respectively, at 0, 5000, 15,000, and 50,000 ppm); however, the study report indicated that since gestation never exceeded 22 days, and since no abnormalities were noted during parturition, this finding was considered not relevant. There were no significant differences noted for any sperm parameter measured for any treated group from the F0 generation.

F0 parents – organ weights: For males, absolute and/or relative adrenals, pituitary, spleen, and thyroid weights were statistically significantly decreased at all test concentrations. Additionally, absolute brain weights were significantly decreased at 15,000 a nd 50,000 ppm, absolute prostate weights were significantly decreased at 5000 and 50,000 ppm, and absolute kidney weights were significantly decreased at 15,000 ppm. For females, absolute adrenal weights were significantly decreased at 50,000 ppm, and absolute pituitary weights were significantly decreased at 15,000 and 50,000 ppm. Relative liver weights were significantly increased in males at 15,000 and 50,000 ppm and in females at 50,000 ppm.

F0 parents – macroscopic and microscopic observations: There were no significant macroscopic observations at any test concentration. Microscopic analyses were not reported in the interim report because they had not yet been performed.

F1 pups: The F1 mean live litter sizes, mean and total numbers of pups born, pup weights, and pupLast updated 1-22-09 survival were unaffected by treatment. The sex ratios (percentages of male pups) on post-natal day 1 were 53, 52, 44, and 41% respectively at 0, 5000, 15,000, and 50,000 ppm, and this was statistically significant at 50,000 ppm with the Fisher’s exact test but not with covariate analysis.

The number of runts (defined as pup weight less than mean pup weight of the control group minus two standard deviations) was statistically significantly decreased at 5000 ppm on LD1 and 4, and at 15,000 and 50,000 ppm on LD4. On LD1, the number of runts (number of litters affected) was 17(7), 4(4)** , 8(5), and 8(5) at 0, 5000, 15,000, and 50,000 ppm respectively (** denotes p<0.01). On LD4, the number of runts (number of litters affected) was 15(6), 0(0)#, 3(3)**, and 4(4)* at 0, 5000, 15,000, and 50,000 ppm respectively (# denotes p<0.001, ** denotes p<0.01, and * denotes p<0.05). As the number of runts (and number of litters affected) was greater in the control group compared to all three treatment groups, and as a decreased number of runts is not clearly an adve rse effect, this effect does not appear to be toxicologically relevant.

There were no test substance-related macroscopic findings at necropsy in F1 pups. At 5000 ppm, absolute and relative thymus weights of pups (both sexes) were significantly decreased and at 50,000 ppm relative thymus weights of female pups were significantly

decreased.

Skeletal examination revealed no significant test substance-related effects. The only findings were as follows:

 0 ppm: One pup with a shortened 13 th rib

 5000 ppm: One pup with a malpositioned 13 th rib and 2 detached ribs; one pup with knobby ribs; 3 pups from 2 litters with a shortened 13 th rib

 15,000 ppm: Two pups (from 2 litters) with knobby ribs; 4 pups (from 4 litters) with

a shortened 13 th rib

 50,000 ppm: One pup with knobby ribs; 2 pups (from 2 litters) with a shortened 13 th

rib

In F1 males examined for preputial separation, there were no statistically significant differences compared with controls. In F1 females, mean time to vaginal opening was statistically significantly delayed at 15,000 and 50,000 ppm (39.36, 40.36, 42.48, and 43.46 days at 0, 5000, 15,000, and 50,000 ppm respectively).

IV. ASSESSMENT OF STUDY QUALITY

The study reportedly followed OECD TG 416. The study appears to have generally followed this guideline with the exception that the inhalation route of exposure was used instead of oral exposure. The study report indicated that the inhalation route was chosen because this is the most likely route of human exposure. Additionally, OECD TG 416 recommends a 2-week mating period, but this study employed a 1-week mating period.Last updated 1-22-09 V. REFERENCES

P-07-0601. Interim report: Inhalatory two-generation reproduction toxicity study with HFO-1234yf in Wistar rats. . Interim report 01. 26 August 2008.

OECD TG 416. Two-generation reproduction toxicity. Adopted 22 January 2001.