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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Oct 2017 to 31 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2001
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline for Testing of Chemicals 412. Subacute Toxicity: 28-Day Study.
Version / remarks:
September 2009
Deviations:
yes
Remarks:
Ony used for the inhalation exposure part.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexafluoropropene
EC Number:
204-127-4
EC Name:
Hexafluoropropene
Cas Number:
116-15-4
Molecular formula:
C3F6
IUPAC Name:
1,1,2,3,3,3-hexafluoroprop-1-ene
Test material form:
gas under pressure: liquefied gas

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han IGS rats (Crl:WI(Han)) (SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 10 weeks females and 9 weeks male upon arrival.
- Weight at study initiation: Group averages were between 200.89 and 208.06 g.
- Fasting period before study: No
- Housing: The animals were housed under conventional conditions in one animal room. No other test system was housed in the same room during the study. During the exposure periods, the rats were individually housed in the exposure unit. When not exposed, animals were housed in macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the rats were housed in groups of four per cage, separated by sex.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) (SDS Special Diets Services, Witham, England)
- Water: ad libitum, tap-water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 29.4 to 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
16 Oct 2017 to 31 Oct 2017

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Remarks:
HEPA filter
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmosphere in 2.2 m3 whole body exposure units (based on the design by Hazleton Systems, Inc., Aberdeen, MD, USA). The test atmosphere was introduced at the top and exhausted at the bottom of the chamber. During exposure, animals were housed individually in Type II Macrolon cages. Each whole body chamber could accommodate 60 cages. Animals were rotated at least twice weekly with respect to their position in the exposure chamber.
- Method of holding animals in test chamber: Not applicable, the animals were exposed via whole body exposure.
- Source and rate of air: Clean air was available as a laboratory provided source of (non-pressurized) filtered air (HEPA filter).
- Method of conditioning air: The test atmospheres were generated by mixing a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, The Netherlands) stream of gaseous test material with a controlled flow of clean air.
- System of generating particulates/aerosols: Not applicable, the test substance is a gas.
- Temperature, humidity, pressure in air chamber: Temperature of 22 ± 3°C and a relative humidity between 30 and 70%. Pressure in the air chamber was not reported.
- Air flow rate: The flow of test atmosphere was controlled using a constant volume valve and was measured in the exhaust of the exposure chamber using a KIMO air velocity sensor (type CTV110-AOD150; KIMO, Emerainville, France). The flow was continuously measured and recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany).
- Air change rate: at least 10 air changes per hour
- Method of particle size determination: Not applicable
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak GMS 810 EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands). Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line. The response of the analyzers was recorded on a PC at one minute intervals using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the analyzers were converted to concentrations by means of calibration graphs.
- Samples taken from breathing zone: Yes

VEHICLE
- Clean air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL VERIFICATION
Prior to the first exposure of the animals, homogeneous distribution of the test material in the exposure chambers was confirmed by analysis of samples taken at five different locations in each exposure chamber. Further details are described in the field ‘Details on exposure’.

CALIBRATION OF EQUIPMENT
Prior to the first exposure, the output of the flame ionization detector of each TCA was calibrated using gas sample bags. To this end, sample bags were filled with accurate (mass flow controlled) volumes of air; mass flow controlled streams of test material were added using a digital timer (Omron H5CX) and a valve system. The mass flow controller was calibrated using a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA) at the flow settings used for filling of the sample bags. Three concentrations were thus prepared (in duplicate) – at approximately 80%, 100% and 120% of the target concentration of each group. A zero calibration was included for each TCA, using clean dry air only. Linear relations were found between the response of the analyzers and the concentration of the test material.
The calibrations were checked weekly during the study. To this end, gas sample bags were prepared at each target concentration as described above, and were subsequently analyzed by the TCA. If the measured concentration deviated more than 5% from the calculated concentration, the calibration check was repeated. If the deviation was more than 5% at the re-check, a complete re-calibration was carried out.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: Animals were caged together until mating occurred.
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
6 hours per exposure
Frequency of treatment:
Daily
Duration of test:
Gestation day 6 up to and including day 20
Doses / concentrationsopen allclose all
Dose / conc.:
50 ppm (nominal)
Remarks:
Group 2 (low dose)
Dose / conc.:
300 ppm (nominal)
Remarks:
Group 3 (mid dose)
Dose / conc.:
900 ppm (nominal)
Remarks:
Group 4 (high dose)
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure levels were selected on the basis of the results of a range finding study with HFP in rats. Concentrations of 450 and 900 ppm showed effects on maternal body weight and food intake and induced clinical signs (piloerection). Based on these results 900 ppm was selected as high concentration in the current study and is anticipated to induce some maternal toxicity. The low concentration of 50 ppm was selected aiming to result in a No Observed Adverse Effect Concentration (NOAEC).
- Animal assignment: Random, in such a way that the animals from the same day of pregnancy were equally distributed over all groups. Females mated by the same male were placed in different groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in the morning hours (pre-dosing) and halfway through the 6-hour exposure period (focussing on breathing abnormalities and restlessness).
- Cage side observations checked in Table 1 in ‘Any other information on materials and methods incl. tables’ were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, after exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the parental female animals was recorded on gestation days (GD) 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION: Yes
- Time schedule for food consumption recordings: The food consumed for each mated female was measured over the periods: GD 0-6, GD 6-9, GD 9-12, GD 12-15, GD 15-18 and GD 18-21.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/per animal/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs weighed: Kidneys, liver, lungs, gravid uterus, empty uterus, ovaries, live fetuses (individually) with corresponding placentas.
- Organs preserved in neutral aqueous phosphate-buffered 4% solution of formaldehyde for possible future histopathological examinations: Lungs, liver, kidneys, gross lesions.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead fetuses, sex of fetuses, number of grossly visible malformed fetuses and fetuses with external abnormalities, gross evaluation of placentas, abnormal tissues of organs in dams.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**). The statistical tests that were selected are presented in the data tables in ‘Any other information on results incl. tables’.
Indices:
For each litter the following data is presented for each group:
- Number of pregnant females
- Number of females with liveborn and (all) stillborn pups
- Post-implantation loss = [(number of implantation sites- number of live fetuses)/number of implantation sites] x 100
- Gestation index = (number of females with live fetuses / number of females pregnant) x 100
- Sex ratio = [(number of live male fetuses / number of live fetuses)] x 100
Historical control data:
-

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
PRE-DOSING SIGNS
The signs observed pre-exposure comprised skin observations (encrustations and sparsely haired areas) that are common in this rat strain, and based on the distribution amongst the groups not related to treatment. One animal in the 50 ppm group had encrustations in the eye, which is considered not related to treatment. However, animals in the high dose group (900 ppm) showed more piloerection (8 animals) and erythema of the ears (2 animals) compared to the controls (0 animals). These effects are considered related to treatment.

SIGNS DURING EXPOSURE
Observation of the animals about halfway through the 6-hour exposure period revealed piloerection in animals of the mid- and high-concentration groups. In animals of the mid concentration group, this finding was observed in all animals on 19, 20, 30 October and 2 November 2017 (and in several animals on 18 and 23 October 2017). In animals of the high concentration group, piloerection was seen in all animals on 19-25, 27, 30, 31 October and 2 November 2017, and in several animals on 17, 18 and 26 October 2017.

POST-DOSING SIGNS
The signs observed post-exposure comprised skin observations (encrustations and sparsely haired areas) that are common in this rat strain, and based on the distribution amongst the groups not related to treatment. However, 1 animal in the mid dose group and 14 animals in the high dosage group showed piloerection.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight or body weight change were observed in the mid, and high concentration groups (Table 1 and 2 in 'Any other information on results incl. tables'). Significantly, but slightly (less than 5% as compared to the control group) lower body weight was observed from gestation day 15 onwards in the 300 ppm group. In the 900 ppm group statistically significant lower body weight of approximately 15% as compared to the control group were observed from gestation day 9 onwards.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Concentration related lowered mean food consumption was observed in the mid and high concentration groups for intervals 6-9 days and later (Table 3 in 'Any other information on results incl. tables').
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A lower full uterus weight (including fetal and placental tissues) was observed in the high concentration group. Similarly, significantly lower empty uterus weight was found in the mid and high concentration groups. No differences were observed for ovary weight (Table 5 in 'Any other information on results incl. tables').

Terminal body weight was significantly lower in the mid and high concentration groups (Table 6 in 'Any other information on results incl. tables'). Mean absolute kidney weight was significantly higher already at the 50 ppm level. When expressed relatively to body weight this was observed only for the 300 and 900 ppm groups. No differences were observed in liver weight. Mean absolute lung weights were higher in the high concentration group. When this parameter was expressed relatively to body weight this was observed already at the 300 ppm level.

No effects on mean placenta weight were observed in all concentration groups (Table 7 in 'Any other information on results incl. tables').
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Several animals in the high concentration group showed discoloration of the kidneys. This was considered to be related to treatment and in combination with the effects on kidney weight considered adverse.
No other treatment-related macroscopic changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of corpora lutea (13.8, 13.4, 14.0 and 13.6 for the control, low, mid and high concentration group, respectively) and the mean number of implantation sites (12.4, 12.4, 12.5, and 11.9 for the control, low, mid and high concentration group, respectively) were comparable in all groups. Similarly, no differences in mean pre-implantation loss were observed (Table 4 in 'Any other information on results incl. tables').
Mean post-implantation loss was comparable over all groups (3.70, 3.86, 5.98 and 4.88 % in the control, low, mid and high concentration groups, respectively, Table 4 in 'Any other information on results incl. tables').
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of early resorptions per litter (0.3, 0.4, 0.7, and 0.6 for the control, low, mid and high concentration groups, respectively) and the number of late resorptions per litter (0.1, 0.0, 0.0, and 0.0 for the control, low, mid and high concentration groups, respectively) were comparable in all groups (Table 4 in 'Any other information on results incl. tables'.).
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
300 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slightly, but statistically significantly lower mean fetal weight was observed in the 300 ppm group (less than 5% lower as compared to the control group, Table 7 in 'Any other information on results incl. tables'). This was considered treatment-related, but not biologically adverse.
Significantly lower mean fetal weight was observed in the 900 ppm groups (more than 15% lower as compared to the control group). This was considered a treatment-related and adverse effect.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
MALFORMATIONS (Table 8 in 'Any other information on results incl. tables')
There were no treatment-related external malformations.

VARIATIONS
Based on the number and distribution of the variations observed there were no treatment-related effects.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
MALFORMATIONS (Table 10 in 'Any other information on results incl. tables')
No treatment-related skeletal malformations were found.

VARIATIONS
Multiple skeletal variations indicative for retardation in ossification were noted. With some exceptions, statistical significance was mostly confined to the high-concentration group.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
MALFORMATIONS (Table 9 in 'Any other information on results incl. tables')
No fetal visceral malformations were observed.

VARIATIONS
Although several visceral observations were made, their incidence was not statistically different in any concentration group.
Based on the above incidences of malformations and variations, it was concluded that visceral examination did not reveal any treatment-related effects.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: Retarded ossification

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
900 ppm (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Actual concentrations in the exposure chambers: The overall average actual concentrations (± standard deviation) of test substance in the test atmospheres, as determined by total carbon analysis, were 50 (± 0.7), 302 (± 3) and 901 (± 16) ppm for the low-, mid- and high-concentration, respectively. These concentrations were close to the respective target concentrations of 50, 300 and 900 ppm.

Table 1. Body weight gestation – Day(s) relative to mating

Sex: Female

Bodyweights

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G1]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G1]

0

6

9

12

15

18

21

0 ppm

Mean

SD

N

202.48

7.68

23

227.67

9.71

23

235.57

10.35

23

247.35

10.61

23

259.85

10.84

23

289.24

13.59

23

318.51

23.26

23

50 ppm

Mean

SD

N

206.72

8.66

22

230.75

9.92

22

237.72

10.90

22

250.30

11.13

22

261.21

11.38

22

289.20

11.96

22

324.82

15.35

22

300 ppm

Mean

SD

N

208.06

7.45

23

231.05

12.35

23

231.04

8.45

23

240.07

9.72

23

248.50 **

10.27

23

277.32 **

11.97

23

307.31 *

14.40

23

900 ppm

Mean

SD

N

200.89

8.40

21

226.90

9.91

21

214.31 **

10.13

21

219.93 **

11.43

21

232.82 **

11.55

21

255.10 **

12.40

21

272.31 **

25.96

21

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks: * = p < 0.05; ** = p < 0.01

 

Table 2. Body weight changes gestation – Day(s) relative to mating

Sex: Female

 

Body wt

change (g)

 

[g]

Body wt

change (g)

 

[g]

Body wt

change (g)

 

[g]

Body wt

change (g)

 

[g1]

Body wt

change (g)

 

[g1]

Body wt

change (g)

 

[g]

0 - 6

6 - 9

9 - 12

12 - 15

15 - 18

18 - 21

0 ppm

Mean

SD

N

25.19

4.71

23

7.89

2.21

23

11.78

2.59

23

12.50

3.42

23

29.39

3.97

23

29.27

14.62

23

50 ppm

Mean

SD

N

24.02

4.85

22

6.98

3.58

22

12.58

2.38

22

10.91

3.85

22

27.99

4.15

22

35.63

7.31

22

300 ppm

Mean

SD

N

23.00

7.72

23

-0.01 **

6.85

23

9.03 **

3.05

23

8.43 **

5.24

23

28.82

5.14

23

29.99

5.49

23

900 ppm

Mean

SD

N

26.01

5.45

21

-12.58 **

5.52

21

5.62 **

4.60

21

12.89

3.62

21

22.29 **

3.03

21

17.21 **

22.91

21

[g] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01

[g1] - Anova & Dunnett: ** = p < 0.01

 

Table 3. Food consumption – Day(s) relative to mating

Sex: Female

Day(s) Relative to Mating (Litter: A)

0 - 6

6 - 9

9 - 12

12 - 15

15 - 18

18 - 21

0 ppm

Mean

SD

N

16.29

1.68

23

16.86

1.50

23

18.12

1.32

23

18.18

1.06

23

21.27

2.23

23

18.63

1.96

23

50 ppm

Mean

SD

N

16.09

1.18

22

16.70

1.52

22

17.88

1.40

22

18.05

2.05

22

19.89

2.38

22

19.01

1.84

22

300 ppm

Mean

SD

N

16.07

1.47

23

14.19 **

1.07

23

15.01 **

1.50

23

16.08 **

1.93

23

18.59 **

1.69

23

17.64

2.40

23

900 ppm

Mean

SD

N

16.57

1.60

21

9.29 **

2.63

21

10.83 **

2.45

21

15.24 **

1.84

21

16.27 **

2.23

21

14.90 **

2.92

21

Dunnett: ** = p < 0.01

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

Table 4. Cesarean section

Sex: Female

0 ppm

50 ppm

300 ppm

900 ppm

Females Mated [f]

N+ve

24

24

24

24

Pregnant at C-section

N+ve

23

22

23

21

Not Pregnant at C-section

N+ve

1

2

1

3

Females Delivered Early

N+ve

0

0

0

0

Dams with no viable Fetuses

N+ve

0

0

0

0

Dams with live Fetuses

N+ve

23

22

23

21

Female Fecundity Index

%

95.8

91.7

95.8

87.5

Gestation Index

%

100.0

100.0

100.0

100.0

Number of Corpora Lutea [k]

Mean

13.8

13.4

14.0

13.6

 

SD

1.8

1.7

1.6

2.0

 

N

23

22

23

21

 

Sum

317

294

322

285

Number of Implantation Sites [k]

Mean

12.4

12.4

12.5

11.9

 

SD

1.6

1.7

1.6

3.0

 

N

23

22

23

21

 

Sum

286

272

287

249

Pre-implantation Loss (%)

Mean

9.49

7.68

10.13

13.30

 

SD

7.47

8.01

12.10

17.84

 

N

23

22

23

21

Number of Live Fetuses [k]

Mean

12.0

11.9

11.7

11.2

 

SD

1.7

2.1

1.7

2.7

 

Sum

275

262

269

235

Post-implantation Loss (%) [k]

Mean

3.70

3.86

5.98

4.88

 

SD

7.34

10.13

9.35

8.20

 

N

23

22

23

21

Number of Dead Fetuses [k]

Mean

0.0

0.0

0.0

0.1

 

SD

0.0

0.0

0.2

0.4

 

Sum

0

0

1

2

Number of Resorptions: Total [k]

Mean

0.5

0.5

0.7

0.6

 

SD

1.0

1.1

1.2

1.1

 

N

23

22

23

21

 

Sum

11

10

17

12

Number of Resorptions: Early [k]

Mean

0.3

0.4

0.7

0.6

 

SD

0.6

1.0

1.2

1.1

 

N

23

22

23

21

 

Sum

8

9

17

12

Number of Resorptions: Late [k]

Mean

0.1

0.0

0.0

0.0

 

SD

0.6

0.2

0.0

0.0

 

N

23

22

23

21

 

Sum

3

1

0

0

Live Male Fetuses [f]

Mean

SD

N

Sum

 

Mean

SD

N

Sum

6.5

2.1

23

150

54.5

5.4

1.7

23

125

45.5

6.1

1.9

22

135

51.7

5.7

1.8

22

126

48.3

5.6

1.5

23

128

47.6

6.1

1.7

23

141

52.4

5.5

2.1

21

115

48.9

5.7

2.2

21

120

51.1

Sex Ratio - Male Fetuses (%)

Live Female Fetuses [f]

Sex Ratio - Female Fetuses (%)

[f] - Chi-Squared & Fisher’s Exact

[k] - Kruskal-Wallis & Wilcoxon

Pre-implantation loss: no. corpora lutea - no. implant sites *100/no. corpora lutea

Post-implantation loss: no. implant sites - no. live fetuses *100/no. implant sites

Sex ratio: no. of live (fe)male fetuses *100/ no. of live fetuses

Female fecundity index: no. of females pregnant * 100 /no. of females mated

Gestation index: no. of females with live fetuses * 100 /no. of females pregnant

 

Table 5. Reproductive organ weights – Day(s) relative to mating

Sex: Female

 

 

 

Gravid

Uterus (g)

 

[g]

Uterus

empty (g)

 

[g1]

Ovaries

 (g)

 

[g1]

21

21

21

0 ppm

Mean

78.6535

4.8273

0.1130

 

SD

10.4029

0.6216

0.0152

 

N

23

22

23

50 ppm

Mean

76.5664

4.5086

0.1147

 

SD

11.3180

0.6201

0.0162

 

N

22

22

22

300 ppm

Mean

74.0945

4.3251 *

0.1153

 

SD

11.1448

0.6968

0.0125

 

N

22

23

23

900 ppm

Mean

63.3548 **

3.8294 **

0.1103

 

SD

13.5118

0.6248

0.0127

 

N

21

21

21

[g] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01

[g1] - Anova & Dunnett: * = p < 0.05; ** = p < 0.01

 

Table 6. Organ weights – Day(s) relative to mating

Sex: Female

Bodyweights

 

 

 

 

 

 

Bodywt

 

(g)

 

[G]

Kidneys

 

(g)

 

[G]

Liver

 

(g)

 

[G1]

Lungs

 

(g)

 

[G1]

Kidneys

relative (g/kg body wgt)

[G]

Liver

relative (g/kg body wgt)

[G]

Lungs

relative (g/kg body wgt)

[G]

21

21

21

21

21

21

21

0 ppm

Mean

SD

N

318.51

23.26

23

1.333

0.130

23

10.133

1.046

23

1.107

0.091

23

4.196

0.405

23

31.87

2.97

23

3.485

0.308

23

50 ppm

Mean

SD

N

324.82

15.35

22

1.438 *

0.127

22

10.423

1.217

22

1.090

0.084

22

4.431

0.401

22

32.05

3.09

22

3.358

0.218

22

300 ppm

Mean

SD

N

307.31 *

14.40

23

1.477 **

0.156

23

10.177

0.730

23

1.129

0.085

23

4.812 **

0.495

23

33.16

2.55

23

3.678 *

0.284

23

900 ppm

Mean

SD

N

272.31 **

25.96

21

1.737 **

0.272

21

9.754

1.352

21

1.220 **

0.092

21

6.424 **

1.084

21

35.98 **

5.25

21

4.526 **

0.607

21

[G] - Kruskal-Wallis & Dunnett on Ranks: * = p < 0.05; ** = p < 0.01

[G1] - Ancova/Anova & Dunnett: ** = p < 0.01

 

Table 7. Placental and fetal weight – Day(s) relative to mating

Sex: Female

 

 

 

 

 

 

Placental wt

(M) mean (g)

 

[g]

Placental wt

(F) mean (g)

 

[g1]

Placental wt

(M+F) mean (g)

 

[g1]

Fetal wt

(M) mean (g)

 

[g]

Fetal wt

(F) mean (g)

 

[g]

Fetal wt

(M+F) mean (g)

 

[g]

21

21

21

21

21

21

0 ppm

Mean

SD

N

0.464

0.048

23

0.436

0.049

23

0.453

0.045

23

5.108

0.232

23

4.848

0.226

23

4.995

0.212

23

50 ppm

Mean

SD

N

0.462

0.051

22

0.422

0.045

22

0.443

0.047

22

4.974

0.250

22

4.694

0.225

22

4.842

0.233

22

300 ppm

Mean

SD

N

0.452

0.047

23

0.433

0.050

23

0.444

0.048

23

4.860 *

0.312

23

4.608 **

0.262

23

4.728 **

0.278

23

900 ppm

Mean

SD

N

0.460

0.067

20

0.442

0.075

21

0.458

0.075

21

4.239 **

0.340

20

4.045 **

0.351

21

4.162 **

0.350

21

[g] - Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[g1] - Anova & Dunnett(Log)

 

Table 8. Fetal external observations

Exam Type: External

 

Number of Fetuses Examined: Number of Litters Examined:

0 ppm

50 ppm

300 ppm

900 ppm

275

23

262

22

269

23

2241

201

Eye

 

 

 

 

 

Eye bulge, Protruding - Variation

Fetuses N(%)

1(0.4)

0(0.0)

0(0.0)

0(0.0)

 

Litters N(%)

1(4.3)

0(0.0)

0(0.0)

0(0.0)

General

 

 

 

 

 

General, Pale - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

1(0.4)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

1(5.0)

General, Small - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.4)

1(0.4)

 

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

1(5.0)

General, Subcutaneous hemorrhage - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

1(0.4)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

1(5.0)

Localized, Subcutaneous hemorrhage - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

1(0.4)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

1(5.0)

Head/Neck

 

 

 

 

 

Head, Small - Variation

Fetuses N(%)

1(0.4)

0(0.0)

0(0.0)

0(0.0)

 

Litters N(%)

1(4.3)

0(0.0)

0(0.0)

0(0.0)

Jaw,lower, Absent - Malformation

Fetuses N(%)

1(0.4)

0(0.0)

0(0.0)

0(0.0)

 

Litters N(%)

1(4.3)

0(0.0)

0(0.0)

0(0.0)

Trunk

 

 

 

 

 

Umbilicus, Hernia - Malformation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.4)

0(0.0)

 

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

0(0.0)

1 = Erroneously no external observations were recorded for one animal

[Fetuses N] - Chi-Squared & Fisher's Exact

[Litters N] - Chi-Squared & Fisher's Exact

 

Table 9. Fetal visceral observations

Exam Type: Visceral

 

 

Number of Fetuses Examined: Number of Litters Examined:

0 ppm

50 ppm

300 ppm

900 ppm

132

23

128

22

129

23

111

21

Eye

 

 

 

 

 

Retina, Fold - Variation

Fetuses N(%)

2(1.5)

6(4.7)

6(4.7)

5(4.5)

 

Litters N(%)

2(8.7)

5(22.7)

5(21.7)

4(19.0)

Liver

 

 

 

 

 

Lobe, Supernumerary - Variation

Fetuses N(%)

1(0.8)

0(0.0)

0(0.0)

0(0.0)

 

Litters N(%)

1(4.3)

0(0.0)

0(0.0)

0(0.0)

Ureter

 

 

 

 

 

Ureter, Bent - Variation

Fetuses N(%)

14(10.6)

8(6.3)

5(3.9)

6(5.4)

 

Litters N(%)

11(47.8)

5(22.7)

5(21.7)

6(28.6)

Ureter, Dilated - Variation

Fetuses N(%)

0(0.0)

1(0.8)

0(0.0)

0(0.0)

 

Litters N(%)

0(0.0)

1(4.5)

0(0.0)

0(0.0)

Ureter, Kinked - Variation

Fetuses N(%)

5(3.8)

7(5.5)

5(3.9)

6(5.4)

 

Litters N(%)

5(21.7)

6(27.3)

3(13.0)

4(19.0)

Urinary Bladder

 

 

 

 

 

Bladder, Distended - Variation

Fetuses N(%)

2(1.5)

3(2.3)

2(1.6)

2(1.8)

 

Litters N(%)

2(8.7)

3(13.6)

2(8.7)

1(4.8)

[Fetuses N] - Chi-Squared & Fisher's Exact

[Litters N] - Chi-Squared & Fisher's Exact

 

Table 10. Fetal skeletal observations

Exam Type: Skeletal

 

Number of Fetuses Examined: Number of Litters Examined:

0 ppm

50 ppm

300 ppm

900 ppm

141

23

133

22

139

23

124

21

Vertebra, caudal

 

 

 

 

 

Caudal bodies, one or two, Unossified - Variation

Fetuses N(%)

30(21.3)

35(26.3)

53(38.4)**

57(46.0)**

 

Litters N(%)

12(52.2)

13(59.1)

16(69.6)

15(71.4)

Caudal bodies, three or more, Unossified - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.7)

0(0.0)

 

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

0(0.0)

Caudal arches, one or two, Incomplete ossification - Variation

Fetuses N(%)

19(13.5)

23(17.3)

16(11.6)

10(8.1)

 

Litters N(%)

8(34.8)

8(36.4)

7(30.4)

6(28.6)

Caudal arches, one or two, Unossified - Variation

Fetuses N(%)

0(0.0)

4(3.0)

0(0.0)

6(4.8)**

 

Litters N(%)

0(0.0)

3(13.6)

0(0.0)

4(19.0)*

Caudal arches, three or more, Unossified - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

1(0.8)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

1(4.8)

Vertebra, cervical

 

 

 

 

 

Cervical bodies, one or two, Incomplete ossification - Variation

Fetuses N(%)

1(0.7)

1(0.8)

2(1.4)

2(1.6)

 

Litters N(%)

1(4.3)

1(4.5)

2(8.7)

2(9.5)

Cervical bodies, one or two, Unossified - Variation

Fetuses N(%)

2(1.4)

2(1.5)

3(2.2)

9(7.3)*

 

Litters N(%)

2(8.7)

2(9.1)

1(4.3)

7(33.3)

Cervical bodies, three or more, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

2(1.6)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

1(4.8)

Cervical bodies, three or more, Unossified - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.7)

12(9.7)**

 

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

9(42.9)**

Vertebra, lumbar

 

 

 

 

 

Lumbar bodies, one or two, Incomplete ossification - Variation

Fetuses N(%)

1(0.7)

0(0.0)

1(0.7)

0(0.0)

 

Litters N(%)

1(4.3)

0(0.0)

1(4.3)

0(0.0)

Lumbar arches, one or two, Incomplete ossification - Variation

Fetuses N(%)

8(5.7)

13(9.8)

3(2.2)

0(0.0)**

 

Litters N(%)

6(26.1)

9(40.9)

2(8.7)

0(0.0)*

Pelvic girdle

 

 

 

 

 

Pubis, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.7)

0(0.0)

Pubis, Incomplete ossification - Variation

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

0(0.0)

Rib

 

 

 

 

 

One Rib, Wavy - Variation

Fetuses N(%)

4(2.8)

2(1.5)

0(0.0)

1(0.8)

 

Litters N(%)

2(8.7)

2(9.1)

0(0.0)

1(4.8)

Two Ribs, Wavy - Variation

Fetuses N(%)

10(7.1)

11(8.3)

3(2.2)

5(4.0)

 

Litters N(%)

5(21.7)

5(22.7)

3(13.0)

3(14.3)

Three or more Ribs, Wavy - Variation

Fetuses N(%)

3(2.1)

3(2.3)

4(2.9)

10(8.1)*

 

Litters N(%)

2(8.7)

3(13.6)

4(17.4)

6(28.6)

Vertebra, sacral

 

 

 

 

 

Sacral bodies, one or two, Unossified - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

3(2.4)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

3(14.3)

Sacral arches, one or two, Incomplete ossification - Variation

Fetuses N(%)

2(1.4)

1(0.8)

0(0.0)

0(0.0)

 

Litters N(%)

1(4.3)

1(4.5)

0(0.0)

0(0.0)

Skull

 

 

 

 

 

Hyoid, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

2(1.5)

0(0.0)

0(0.0)

 

Litters N(%)

0(0.0)

1(4.5)

0(0.0)

0(0.0)

Interparietal, Incomplete ossification - Variation

Fetuses N(%)

5(3.5)

13(9.8)*

13(9.4)

20(16.1)**

 

Litters N(%)

4(17.4)

8(36.4)

8(34.8)

8(38.1)

Mandible, Hole - Variation

Fetuses N(%)

4(2.8)

4(3.0)

4(2.9)

6(4.8)

 

Litters N(%)

3(13.0)

3(13.6)

2(8.7)

3(14.3)

Mandible, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

1(0.8)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

1(4.8)

Nasal, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.7)

0(0.0)

 

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

0(0.0)

Parietal, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

8(6.0)**

7(5.0)**

17(13.7)**

 

Litters N(%)

0(0.0)

4(18.2)

4(17.4)

4(19.0)

Supraoccipital, Hole - Variation

Fetuses N(%)

1(0.7)

3(2.3)

1(0.7)

2(1.6)

 

Litters N(%)

1(4.3)

3(13.6)

1(4.3)

2(9.5)

Supraoccipital, Incomplete ossification - Variation

Fetuses N(%)

5(3.5)

21(15.8)**

18(12.9)**

28(22.6)**

 

Litters N(%)

4(17.4)

12(54.5)*

10(43.5)

12(57.1)*

Frontal, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

4(3.0)

5(3.6)*

12(9.7)**

 

Litters N(%)

0(0.0)

3(13.6)

2(8.7)

4(19.0)

Squamosal, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

2(1.6)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

2(9.5)

Zygomatic arch, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

1(0.8)

3(2.2)

0(0.0)

 

Litters N(%)

0(0.0)

1(4.5)

1(4.3)

0(0.0)

Sternebra

 

 

 

 

 

One Sternebra, Bipartite ossification - Variation

Fetuses N(%)

1(0.7)

0(0.0)

1(0.7)

0(0.0)

 

Litters N(%)

1(4.3)

0(0.0)

1(4.3)

0(0.0)

One Sternebra, Dumbbell ossification - Variation

Fetuses N(%)

0(0.0)

0(0.0)

0(0.0)

2(1.6)

 

Litters N(%)

0(0.0)

0(0.0)

0(0.0)

2(9.5)

One Sternebra, Incomplete ossification - Variation

Fetuses N(%)

71(50.4)

66(49.6)

78(56.1)

51(41.1)

 

Litters N(%)

23(100.0)

21(95.5)

22(95.7)

18(85.7)

One Sternebra, Irregularly ossified - Variation

Fetuses N(%)

1(0.7)

1(0.8)

0(0.0)

0(0.0)

 

Litters N(%)

1(4.3)

1(4.5)

0(0.0)

0(0.0)

One Sternebra, Irregularly shaped - Variation

Fetuses N(%)

21(14.9)

19(14.3)

21(15.1)

17(13.7)

 

Litters N(%)

13(56.5)

13(59.1)

13(56.5)

11(52.4)

Two Sternebrae, Dumbbell ossification - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.7)

0(0.0)

 

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

0(0.0)

Two Sternebrae, Incomplete ossification - Variation

Fetuses N(%)

7(5.0)

6(4.5)

10(7.2)

28(22.6)**

 

Litters N(%)

4(17.4)

6(27.3)

9(39.1)

13(61.9)**

Two Sternebrae, Unossified - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.7)

0(0.0)

Two Sternebrae, Unossified - Variation

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

0(0.0)

Two Sternebrae, Irregularly shaped - Variation

Fetuses N(%)

14(9.9)

9(6.8)

9(6.5)

16(12.9)

 

Litters N(%)

5(21.7)

8(36.4)

7(30.4)

9(42.9)

Three or more Sternebrae, Incomplete ossification - Variation

Fetuses N(%)

1(0.7)

0(0.0)

0(0.0)

3(2.4)

 

Litters N(%)

1(4.3)

0(0.0)

0(0.0)

2(9.5)

Three or more Sternebrae, Irregularly shaped - Variation

Fetuses N(%)

5(3.5)

4(3.0)

12(8.6)

7(5.6)

 

Litters N(%)

5(21.7)

4(18.2)

9(39.1)

4(19.0)

Vertebra, thoracic

 

 

 

 

 

Thoracic bodies, one or two, Asymmetric ossification - Variation

Fetuses N(%)

1(0.7)

0(0.0)

2(1.4)

0(0.0)

 

Litters N(%)

1(4.3)

0(0.0)

2(8.7)

0(0.0)

Thoracic bodies, one or two, Incomplete ossification - Variation

Fetuses N(%)

7(5.0)

18(13.5)

16(11.5)

11(8.9)

 

Litters N(%)

6(26.1)

11(50.0)

9(39.1)

4(19.0)

Thoracic bodies, three or more, Incomplete ossification - Variation

Fetuses N(%)

2(1.4)

3(2.3)

1(0.7)

1(0.8)

 

Litters N(%)

2(8.7)

3(13.6)

1(4.3)

1(4.8)

Supernumerary rib

 

 

 

 

 

Thoracolumbar, Full - Variation

Fetuses N(%)

7(5.0)

8(6.0)

1(0.7)

10(8.1)

 

Litters N(%)

5(21.7)

5(22.7)

1(4.3)

8(38.1)

Thoracolumbar, Long - Variation

Fetuses N(%)

4(2.8)

1(0.8)

0(0.0)

0(0.0)

 

Litters N(%)

2(8.7)

1(4.5)

0(0.0)

0(0.0)

Thoracolumbar, Short - Variation

Fetuses N(%)

81(57.4)

69(51.9)

75(54.0)

64(51.6)

 

Litters N(%)

20(87.0)

17(77.3)

23(100.0)

19(90.5)

Forelimb, phalanges proximal

 

 

 

 

 

1-4 Digits, Incomplete ossification - Variation

Fetuses N(%)

53(37.6)

58(43.6)

69(49.6)

42(33.9)

 

Litters N(%)

20(87.0)

21(95.5)

18(78.3)

16(76.2)

1-4 Digits, Unossified - Variation

Fetuses N(%)

36(25.5)

47(35.3)

47(33.8)

62(50.0)**

 

Litters N(%)

17(73.9)

17(77.3)

18(78.3)

18(85.7)

5-8 Digits, Incomplete ossification - Variation

Fetuses N(%)

4(2.8)

1(0.8)

0(0.0)

3(2.4)

 

Litters N(%)

2(8.7)

1(4.5)

0(0.0)

2(9.5)

5-8 Digits, Unossified - Variation

Fetuses N(%)

2(1.4)

3(2.3)

9(6.5)*

15(12.1)**

 

Litters N(%)

1(4.3)

3(13.6)

5(21.7)

6(28.6)

Forelimb, phalanges distal

 

 

 

 

 

1-5 Digits, Incomplete ossification - Variation

Fetuses N(%)

14(9.9)

22(16.5)

19(13.7)

19(15.3)

 

Litters N(%)

11(47.8)

11(50.0)

10(43.5)

9(42.9)

1-5 Digits, Unossified - Variation

Fetuses N(%)

12(8.5)

14(10.5)

17(12.2)

23(18.5)

 

Litters N(%)

8(34.8)

9(40.9)

13(56.5)

10(47.6)

6-10 Digits, Incomplete ossification - Variation

Fetuses N(%)

3(2.1)

3(2.3)

8(5.8)

5(4.0)

 

Litters N(%)

2(8.7)

3(13.6)

6(26.1)

4(19.0)

6-10 Digits, Unossified - Variation

Fetuses N(%)

2(1.4)

2(1.5)

3(2.2)

4(3.2)

 

Litters N(%)

2(8.7)

2(9.1)

3(13.0)

3(14.3)

Forelimb, metacarpal

 

 

 

 

 

1-2 Metacarpals, Incomplete ossification - Variation

Fetuses N(%)

0(0.0)

2(1.5)

1(0.7)

10(8.1)**

 

Litters N(%)

0(0.0)

2(9.1)

1(4.3)

5(23.8)*

1-2 Metacarpals, Unossified - Variation

Fetuses N(%)

0(0.0)

0(0.0)

1(0.7)

0(0.0)

 

Litters N(%)

0(0.0)

0(0.0)

1(4.3)

0(0.0)

Hindlimb, metatarsal

 

 

 

 

 

1-2 Metatarsals, Incomplete ossification - Variation

Fetuses N(%)

8(5.7)

10(7.5)

27(19.4)**

15(12.1)

 

Litters N(%)

4(17.4)

5(22.7)

12(52.2)*

9(42.9)

1-2 Metatarsals, Unossified - Variation

Fetuses N(%)

1(0.7)

1(0.8)

10(7.2)**

53(42.7)**

 

Litters N(%)

1(4.3)

1(4.5)

5(21.7)

15(71.4)**

Hindlimb, phalanges distal

 

 

 

 

 

6-10 Digits, Incomplete ossification - Variation

Fetuses N(%)

11(7.8)

5(3.8)

15(10.8)

19(15.3)

 

Litters N(%)

5(21.7)

2(9.1)

7(30.4)

4(19.0)

6-10 Digits, Unossified - Variation

Fetuses N(%)

5(3.5)

3(2.3)

7(5.0)

9(7.3)

 

Litters N(%)

3(13.0)

2(9.1)

4(17.4)

6(28.6)

1-5 Digits, Incomplete ossification - Variation

Fetuses N(%)

6(4.3)

7(5.3)

17(12.2)

10(8.1)

 

Litters N(%)

5(21.7)

6(27.3)

9(39.1)

6(28.6)

1-5 Digits, Unossified - Variation

Fetuses N(%)

6(4.3)

7(5.3)

14(10.1)

8(6.5)

 

Litters N(%)

5(21.7)

5(22.7)

10(43.5)

6(28.6)

Hindlimb, phalanges proximal

 

 

 

 

 

1-4 Digits, Incomplete ossification - Variation

Fetuses N(%)

50(35.5)

36(27.1)

32(23.0)*

6(4.8)**

 

Litters N(%)

17(73.9)

16(72.7)

13(56.5)

4(19.0)**

1-4 Digits, Unossified - Variation

Fetuses N(%)

43(30.5)

39(29.3)

34(24.5)

11(8.9)**

 

Litters N(%)

19(82.6)

20(90.9)

15(65.2)

8(38.1)**

5-8 Digits, Incomplete ossification - Variation

Fetuses N(%)

16(11.3)

17(12.8)

17(12.2)

6(4.8)

 

Litters N(%)

8(34.8)

11(50.0)

9(39.1)

5(23.8)

5-8 Digits, Unossified - Variation

Fetuses N(%)

41(29.1)

52(39.1)

83(59.7)**

105(84.7)**

 

Litters N(%)

15(65.2)

17(77.3)

21(91.3)

20(95.2)*

[Fetuses N] - Chi-Squared & Fisher's Exact: * = p < 0.05; ** = p < 0.01

[Litters N] - Chi-Squared & Fisher's Exact: * = p < 0.05; ** = p < 0.01

Applicant's summary and conclusion

Conclusions:
In this GLP compliant OECD 414 study, the observed effects on body weight, feed intake and kidney weight (supported by macroscopic observations in the 900 ppm group) were considered related to treatment and adverse. Therefore, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was placed at 300 ppm. The effect on fetus weight, accompanied by a retardation in ossification observed in the 900 ppm group was considered adverse. Therefore NOAEC for developmental toxicity was placed at 300 ppm.
Executive summary:

The objective of this GLP compliant OECD 414 study was to provide data on the possible effects of hexafluoropropene on pregnant female Wistar rats, and on the development of the embryo and fetus. The test material was administered by inhalation (whole body exposure) to groups of 24 mated females from gestation day (GD) 6 up to and including GD 20 at concentrations of 50, 300 and 900 ppm. Verification of the target concentrations were performed throughout exposure. Maternal rats were observed for manifestation of clinical signs before, during and after exposure. Body weights of the parental female animals was recorded on gestation days (GD) 0, 6, 9, 12, 15, 18 and 21. Food consumed for each mated female was measured. The females were killed by exsanguination after isofluorane anaesthesia on GD 21 and examined for gross abnormalities, organ weights and uterine content. Fetuses were examined for external alterations and sacrificed by hypothermia. Subsequently, approximately half of the fetuses of each litter were examined for soft tissue anomalies and the other half were examined for skeletal abnormalities.

The overall average actual concentrations (± standard deviation) of hexafluoropropene in the test atmospheres, as determined by total carbon analysis, were 50 (± 0.7), 302 (± 3) and 901 (± 16) ppm for the low-, mid- and high-concentration, respectively. These concentrations were close to the respective target concentrations of 50, 300 and 900 ppm. At gestation day 21 caesarean section was performed, dams were examined macroscopically and reproductive organs, liver and kidney were weighed. Fetuses and placentas were weighed and fetuses were examined externally. The exposure to the test material was well tolerated at low and mid concentration and did not induce treatment-related changes in general condition. Females in the high concentration group frequently showed piloerection. Statistically significantly reduced body weight and body weight gain was observed in the females of the mid and high concentration group. Mean body weight at the end of gestation in the mid concentration group was less than 5% lower as compared to the control group. This was considered a slightly lower body weight and not considered to be adverse. Mean body weight at the end of gestation in the high concentration group was 15% lower as compared to the control group. This was considered an adverse and treatment-related effect. Concentration related lowered mean food consumption was observed in the mid and high concentration groups for intervals 6-9 days and later. Uterus weight for both full and empty uterus was lower in the high concentration group, which could be related to maternal toxicity. Organ weights revealed an increase in weight of the kidneys, visible already at the 50 ppm concentration. Macroscopic pathologic observation of the kidneys showed enlargement and discoloration in the 900 ppm group. In combination with the effects on kidney weight this was considered a treatment-related and adverse effect.

The slightly lower body weight and feed intake observed in the 300 ppm group were considered related to treatment, but in view of the severity considered not adverse. The observed effects on body weight, feed intake and kidney weight (supported by macroscopic observations in the 900 ppm group) were considered related to treatment and adverse. Therefore, the No Observed Adverse Effect Concentration(NOAEC)for maternal toxicity was placed at 300 ppm.

Mean fetus weight was statistically significantly lower in the mid and high concentration groups. A concentration-dependent effect was observed with less than 5% lower fetus weight in the 300 ppm group and more than 15% lower fetus weight in the 900 ppm group as compared to the control group. Furthermore, a number of skeletal observations indicative of retardation in ossification were noted. With some exceptions, statistical significance was mostly confined to the high-concentration group. The retardation in ossification is considered to be related to the lower fetus weight observed.

The slight effect on fetus weight and ossification in the 300 ppm group was considered related to treatment, but in view of the severity were not considered to be biologically relevant and, therefore, not adverse. The effect on fetus weight, accompanied by a retardation in ossification observed in the 900 ppm group was considered adverse. Therefore NOAEC for developmental toxicity was placed at 300 ppm.