Hexafluoropropene

Developmental toxicity / teratogenicity

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm (nominal)

Remarks:

Group 2 (low dose)

Doses / concentrations 2

Dose / conc.:

300 ppm (nominal)

Remarks:

Group 3 (mid dose)

Doses / concentrations 3

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.