EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 DECISIONS 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: C3H2F4

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 = - 152.2oC (Beilstein); BP = - 28.3oC 

(Beilstein); VP = 88 psia at 21.1o

C (Meas) = 4551 torr, 206 psia at 54.4oC (Meas) = 10653 torr 

WS = 198.2 mg/L at 24oC (Meas); Log Kow: 2.15 (est) 

Exposure Routes of Concern: Worker and Consumer via Inhalation 

II. HEALTH ASSESSMENT SUMMARY 

Background This is the second RAD disposition meeting on P07-601. At the first disposition 

meeting, there were questions about the rat developmental toxicity study and whether the effects 

seen were significant. It was decided to evaluate a satellite on a 2 generation study being run by 

the submitter to address this issue. The submitter 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 placental weights and “small 

fetuses” has any significance. If the ribs are normal in the weanling satellite to this 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. 

Page 1 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 (attached) of the interim 

report (1st generation only) of inhalation 2-generation reproductive toxicity study in rats and 

recommends NOAELS for the following observations that may have been significant. 

1. Significantly decreased sex ratio 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 weights 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 considered compound related. 

3. Significantly delayed mean time to vaginal opening in F1 females at 15,000 and 50,000 

ppm. The NOAEL for this effect was 5,000 ppm. 

4. Some minor rib variations were noted but the frequency was low, not statistically 

significant and not dose related. Therefore the changes are not considered compound 

related. 

NOAEL FOR MOE CALCUALTIONS INVOLVING MULTIPLE EXPOSURES: At the 

first RAD disposition meeting, the rabbit developmental 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 will be used in MOE calculations involving 

multiple exposure scenarios. This type of exposure is generally inappropriate for calculation 

MOEs from single exposures, especially 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 INVOLVING SINGLE EXPOSURES: [ 

 ] conducted a 4 hour acute toxicity test of the PMN chemical, nose only, in Sprague 

Dawley rats involving 5 males and 5 females at each dose. Exposures were to 201,600 ppm and 

405,800 ppm. Oxygen was maintained at 19%. Animals were kept for 14 days after exposure 

and gross necropsy performed. No rats died but at necropsy “grey discoloured lungs” were seen 

in 1 female and 1 male of the low dose group and 1 female and 3 males of the high dose group. 

Authors could not discount the lung effects as compound related since there was no control 

group. In the absence of any evidence to the contrary, 201,600 ppm will be considered a 

LOAEL. 

Page 2 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 week of two weeks. Analytically measured 

concentrations were 4,990, 19,599, and 51,690 ppm. Pathology and histopathology did not 

reveal any compound related effects. The only effect observed was 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 range. If no effects were seen 

with multiple exposures to 51,690 ppm, then a single exposure for 6 hours would also be without 

effect. Therefore, 51,690 ppm will be used as the NOAEL for acute exposure calculations of the 

MOE. 

HUMAN EQUIVALENT CONCENTRATION (HEC) DETERMINATION The Human 

Equivalent Concentration is the exposure to humans 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 Concentration (HEC) for the NOAEL for lethality 

in a rabbit developmental toxicity study. The NOAEL 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 showed lethality was 5,500 ppm with the first death 

occurring on GD13 after 8 exposures. Blood levels of the PMN are a better measure of dose 

than external exposure concentrations. The submitter 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 partition 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 were integrated into a model to relate exposure 

to blood dose levels [ ]. Steady state is reached within minutes in animals and humans. 

Exposure values vs blood concentrations 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** 

Page 3 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 exposure 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 exposure 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 give 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 exposure model. However, since blood levels seem to 

be directly proportional to exposure levels in the 400 ppm to 80,000 ppm range it is reasonable to 

use this relationship to predict 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 4,000 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 single 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 (AUC) CONSIDERATIONS 

The submitter uses AUC considerations to predict short duration high level exposures. The 

4,000 ppm blood levels in the rabbit developmental 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 levels 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 this type of calculation 

would indicate an adequate MOE. While the conclusion might have some merit (see below) 

there are still uncertainties with this type of extrapolation. The results of the rabbit 

developmental toxicity studies raise some interesting questions. The exposures 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 

 *estimated 

Page 4 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 that 4,000 ppm is a NOAEL for lethality in the rabbit and that AUC 

calculations can be done for high level, short exposure durations but there is a moderate degree 

of uncertainty regarding the conclusions one can draw about safety. 

III. RISK ASSESSMENT – HEALTH 

Toxicity data: Rabbit developmental toxicity study with 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 for 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. This is a reasonable scientific compromise given 

the uncertainties involved. Other uncertainties involve extrapolating 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 within the human population. These 

factors each comprise two sub-factors – a pharmacokinetic component and a pharmacodynamic 

component which by convention are considered roughly equal. The pharmacokinetic component 

Page 5 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 differences in which species and individuals respond 

to the compound in a toxic response. The HEC is calculated using pharmacokinetic modeling 

which removes the uncertainties of the pharmacokinetic component when extrapolating from 

animals to humans. For this reason the interspecies uncertainty should be reduced from 10 to 

approximately 3. The total MOE would then become 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 Exposure Model (MCCEM) USEPA modeling: compact car 

of 2.5 m3

 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; average event concentration, 86,328 mg/m3 (18,516 ppm); 

Peak concentration, 1.75 E5 mg/m3 (37,500 ppm) 

Number of U. S. exposures/yr: 164 to 3,114 from leak 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 their model. A TWA of 18,516 ppm 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/m3 (556 ppm); Peak 

concentration, 5.2 E3 mg/m3 (1,100 ppm); 

Number of U. S. exposures/yr: 7 to 1,157 from 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 Scenarios

Page 6 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 their model. A TWA of 3,264 ppm 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 their model. A TWA of 45,000 ppm 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 obtain 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 showing any chemical-related effects. 

Page 7 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 scenario (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 exposure 

Page 8 of 19
EPA SANITIZED 

TI: Maggie Johnson 564-8924 RAD 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 rat data because there were no single exposure 

data on the pregnant rabbit, the most susceptible group. There is some uncertainty about whether 

toxicity would be seen in a rabbit from a single exposure to 50,000 ppm. For that reason a rabbit 

acute toxicity study can be conducted to address this question. Rabbits should be exposed for 1 

hour (acute exposure scenarios are for 5 to 30 minutes) 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 (1st Generation Only) of Inhalation 2-generation Reproductive 

Toxicity Study in Rats for P-07-0601 

REFERENCES: 

Review of Interim Report (1st Generation Only) of Inhalation 2-generation Reproductive 

Toxicity Study in Rats for P-07-0601. 

Page 9 of 19
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 were

also 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 periods 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; gross evaluation of the placentas. The following

reproductive 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 low-, 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 food

Page 10 of 19
Last 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 seen in the high-concentration group during GD 6

through 19. No clear concentration-related effects 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 fecundity or gestation indices, numbers of

implantations, pre- and post-implantation losses, resorptions, live fetuses, or mean sex ratio at

any 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 mid-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 group, this effect was not considered an adverse

treatment-related effect. There were no effects 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 effect, 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 weights 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 considered

Page 11 of 19
1 SRC consulted EPA developmental toxicologist Jennifer Seed (EPA RAD), 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 experts 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’s

review, 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]

Page 12 of 19
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 were

also 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 inhalation toxicity study with HFO-1234yf in rats. 

 April 17, 2007.

Kast, A. 1994. Wavy Ribs. A Reversible Pathologic Finding in Rat Fetuses. Exp Toxicol

Page 13 of 19
Last 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.

Page 14 of 19
Last updated 1-22-09 

REVIEW OF INTERIM REPORT (1st GENERATION ONLY) OF INHALATION 2-

GENERATION REPRODUCTIVE TOXICITY 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 

(AHFO-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 parents1

 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, statistically 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. 

Page 15 of 19
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) through day 19 of gestation, and then again via 

whole-body inhalation (6 hours/day) from lactation 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 onwards. 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 females, the following organs were preserved: 

adrenals, brain, epididymides, kidneys, liver, ovaries including oviduct, pituitary gland, prostate, 

seminal vesicles and coagulating glands, spleen, 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. 

Page 16 of 19
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 weeks 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 consumption (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 finding 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 and 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 pup 

Page 17 of 19
Last 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 adverse 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 13th rib 

 5000 ppm: One pup with a malpositioned 13th rib and 2 detached ribs; one pup with 

knobby ribs; 3 pups from 2 litters with a shortened 13th rib 

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

a shortened 13th rib 

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

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. 

Page 18 of 19
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. 

Page 19 of 19